RESUMO
Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.
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Proteínas de Escherichia coli , Sequência de Bases , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Nitratos/metabolismo , Óperon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.
Assuntos
Técnicas de Cultura Celular por Lotes , Escherichia coli , Aldeído Liases/genética , Aldeído Liases/metabolismo , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Fosfatos/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/metabolismoRESUMO
Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions.
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Adesinas Bacterianas/biossíntese , Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Exposição Ambiental , Escherichia coli/metabolismo , Adesinas Bacterianas/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Plásticos/química , Politetrafluoretileno/química , Aço Inoxidável/química , Propriedades de SuperfícieRESUMO
Carbon Storage Regulator A (CsrA) is an RNA binding protein that acts as a global regulator of diverse genes. Using a combination of genetics and biochemistry we show that CsrA binds directly to the 5' end of the transcript encoding AcrAB. Deletion of csrA or mutagenesis of the CsrA binding sites reduced production of both AcrA and AcrB. Nucleotide substitutions at the 5' UTR of acrA mRNA that could potentially weaken the inhibitory RNA secondary structure, allow for more efficient translation of the AcrAB proteins. Given the role of AcrAB-TolC in multi-drug efflux we suggest that CsrA is a potential drug target.
Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Regiões 5' não Traduzidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Genéticos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.
Assuntos
Técnicas de Cultura Celular por Lotes , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Biomassa , Reatores Biológicos/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , Plasmídeos/análise , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Temperatura , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/isolamento & purificaçãoRESUMO
BACKGROUND: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. RESULTS: Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. CONCLUSIONS: Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.
Assuntos
Biofilmes , Enzimas/metabolismo , Engenharia Genética/métodos , Biocatálise , Catálise , Cromatografia Líquida , Enzimas/biossíntese , Enzimas/genética , Espectrometria de Massas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recombinant protein production in bacterial hosts is a commercially important process in the pharmaceutical industry. Optimisation of such processes is of critical importance for process productivity and reproducibility. Here, flow cytometry methods were developed to assess characteristics of bacteria during two process steps that are infrequently studied: agar plate culture and liquid culture set-up. During storage on agar plates, three discrete populations of varying green fluorescence intensity were observed along with a progressive shift of cells from the high green fluorescence population to an intermediate green fluorescence population, observed to be due formation of amyloid inclusion bodies. The dynamics of cellular fluorescence and scatter properties upon setup of liquid cultures were also assessed. These methods have the potential to improve the development of fermentation set-up, a currently little-understood area.
Assuntos
Escherichia coli/química , Escherichia coli/metabolismo , Citometria de Fluxo/métodos , Proteínas Recombinantes/metabolismo , Ágar , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Production of recombinant proteins is an industrially important technique in the biopharmaceutical sector. Many recombinant proteins are problematic to generate in a soluble form in bacteria as they readily form insoluble inclusion bodies. Recombinant protein solubility can be enhanced by minimising stress imposed on bacteria through decreasing growth temperature and the rate of recombinant protein production. In this study, we determined whether these stress-minimisation techniques can be successfully applied to industrially relevant high cell density Escherichia coli fermentations generating a recombinant protein prone to forming inclusion bodies, CheY-GFP. Flow cytometry was used as a routine technique to rapidly determine bacterial productivity and physiology at the single cell level, enabling determination of culture heterogeneity. We show that stress minimisation can be applied to high cell density fermentations (up to a dry cell weight of >70 g L(-1)) using semi-defined media and glucose or glycerol as carbon sources, and using early or late induction of recombinant protein production, to produce high yields (up to 6 g L(-1)) of aggregation-prone recombinant protein in a soluble form. These results clearly demonstrate that stress minimisation is a viable option for the optimisation of high cell density industrial fermentations for the production of high yields of difficult-to-produce recombinant proteins, and present a workflow for the application of stress-minimisation techniques in a variety of fermentation protocols.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Escherichia coli/genética , Fermentação , Glucose/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
Systematic development of a temperature-controlled isocratic process for one-column low-salt hydrophobic interaction chromatography (HIC) of proteins employing a travelling cooling zone reactor (TCZR) system, is described. Batch binding and confocal scanning microscopy were employed to define process conditions for temperature-reversible binding of bovine serum albumin (BSA) which were validated in pulse-response temperature switching HIC experiments, before transferring to TCZR-HIC. A thin-walled stainless-steel column mounted with a movable assembly of copper blocks and Peltier elements (travelling cooling zone, TCZ) was used for TCZR-HIC. In pulse-response TCZR-HIC, 12 TCZ movements along the column desorbed 86.3% of the applied BSA monomers in 95.3% purity depleted >6-fold in 2-4 mers and nearly 260-fold in higher molecular weight (HMW) species. For continuous TCZR-HIC, the TCZ was moved 49-58 times during uninterrupted loading of BSA feeds at 0.25, 0.5 or 1 mg·mL-1. Each TCZ movement generated a sharp symmetrical elution peak. In the best case, (condition 1: 0.25 mg·mL-1 BSA; >17 mg BSA applied per mL of bed) the height of TCZ elution peaks approached pseudo-steady midway through the loading phase with no rise in baseline UV280 signal between peaks. Peak composition remained constant averaging 94.4% monomer, 5.6% 2-4 mers and <0.05% HMW. Monomers were recovered in quantitative yield depleted >3.1 fold in 2-4 mers and 92-fold in HMW species cf. the feed (63.6% monomers, 21.8% 2-4 mers, 14.6% HMW). However, increasing the BSA concentration to 1 mg·mL-1 (condition 2) or employing a fouled HIC column with 0.5 mg·mL-1 BSA (condition 3) compromised monomer purification performance.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Soroalbumina Bovina , Temperatura , Soroalbumina Bovina/química , Cromatografia Líquida/métodos , Animais , BovinosRESUMO
The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development.
Assuntos
Produtos Biológicos , Proteínas Periplásmicas , Escherichia coli/genética , Periplasma , Proteínas RecombinantesRESUMO
As the production of graphene-based nanomaterials such as GO is increasing, it is expected that a large amount of GO waste will be generated. The environment (i.e., soil and aquatic systems) will be amongst the final repositories of these wastes which means important natural microbial communities in such environments will be at risk of GO exposure. However, little is known about how these communities respond to environmental stresses in synergy with the presence of GO. In this study, the effect of three different stress conditions: temperature (5, 25 and 40 °C); pH (5 to 9) and osmotic stress (51, 219 and 320 mM NaCl) in addition to GO treatment was investigated on the viability and physiology of biofilms and planktonic cells of soil bacterium P. putida. It was found that planktonic cells were more resistant to GO alone compared to biofilms. However, the cells were sensitive to GO when exposed to pH or osmotic stresses. Temperature was not found to influence the survival of biofilm with or without exposure to GO. However, low pH caused a reduction in colony-forming units (CFU) at pHs 5 and 6 for the pre-treated samples, while biofilms at pH 7-9 did not show any decrease. Interestingly, the post-treatment of planktonic cells or biofilms with GO showed a significant reduction in CFU at all pH ranges. The effect of higher osmotic stress in combination with GO resulted in a significant reduction in biofilms. These results show that the effect of stresses naturally occurring in the environment can be affected and changed when in combination with GO and can potentially affect the balance of natural biofilms.
RESUMO
Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.
RESUMO
The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.
Assuntos
Biotecnologia/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Coloração e Rotulagem/métodos , Microbiologia Ambiental , Manipulação de Alimentos/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The intrinsic resilience of biofilms to environmental conditions makes them an attractive platform for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiology and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using a combination of controlled radical polymerization and dynamic covalent chemistry, we prepare a set of synthetic polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties. We then demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in MC4100, a strain of Escherichia coli that forms biofilms poorly, with aromatic and heteroaromatic moieties leading to the best performing polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expression of the adhesin curli. In the presence of selected polymers, MC4100 can reach levels of biomass production and curli expression similar or higher than PHL644, demonstrating that synthetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Biocatálise , Biofilmes , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Polímeros/farmacologiaRESUMO
We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. Experiments using the expression of ß-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1ß and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production.
Assuntos
Proteínas de Escherichia coli , Ureia , Humanos , Ureia/farmacologia , Ureia/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteus mirabilis/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
INTRODUCTION: Congenital anomalies affect over 2% of pregnancies. Surgical advances have reduced mortality and improved survival for patients with congenital anomalies potentially requiring surgical (CAPRS) intervention. However, our understanding of aetiology, diagnostic methods, optimal management, outcomes and prognostication is limited. Existing birth cohorts have low numbers of individual heterogenous CAPRS. The Surgical Paediatric congEnital Anomalies Registry with Long term follow-up (Surgical-PEARL) study aims to establish a multicentre prospective fetal, child and biological parent cohort of CAPRS. METHODS AND ANALYSIS: From 2022 to 2027, Surgical-PEARL aims to recruit 2500 patients with CAPRS alongside their biological mothers and fathers from up to 15 UK centres. Recruitment will be antenatal or postnatal dependent on diagnosis timing and presentation to a recruitment site. Routine clinical data including antenatal scans and records, neonatal intensive care unit (NICU) records, diagnostic and surgical data and hospital episode statistics will be collected. A detailed biobank of samples will include: parents' blood and urine samples; amniotic fluid if available; children's blood and urine samples on admission to NICU, perioperatively or if the child has care withdrawn or is transferred for extracorporeal membrane oxygenation; stool samples; and surplus surgical tissue. Parents will complete questionnaires including sociodemographic and health data. Follow-up outcome and questionnaire data will be collected for 5 years. Once established we will explore the potential of comparing findings in Surgical-PEARL to general population cohorts born in the same years and centres. ETHICS AND DISSEMINATION: Ethical and health research authority approvals have been granted (IRAS Project ID: 302251; REC reference number 22/SS/0004). Surgical-PEARL is adopted onto the National Institute for Health Research Clinical Research Network portfolio. Findings will be disseminated widely through peer-reviewed publication, conference presentations and through patient organisations and newsletters. TRIAL REGISTRATION NUMBER: ISRCTN12557586.
Assuntos
Anormalidades Congênitas , Cuidado Pré-Natal , Diagnóstico Pré-Natal , Criança , Feminino , Humanos , Recém-Nascido , Gravidez , Unidades de Terapia Intensiva Neonatal , Estudos Multicêntricos como Assunto , Estudos Prospectivos , Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/cirurgia , PerinatologiaRESUMO
Initial work to generate physically robust biofilms for biocatalytic applications revealed that Escherichia coli K-12 can form a floating biofilm at the air-liquid interface, commonly referred to as a pellicle. Unlike other species where pellicle formation is well-characterised, such as Bacillus subtilis, there are few reports of E. coli K-12 pellicles in the literature. In order to study pellicle formation, a growth model was developed and pellicle formation was monitored over time. Mechanical forces, both motility and shaking, were shown to have effects on pellicle formation and development. The role and regulation of curli, an amyloid protein adhesin critical in E. coli K-12 biofilm formation, was studied by using promoter-green fluorescent protein reporters; flow cytometry and confocal laser scanning microscopy were used to monitor curli expression over time and in different locations. Curli were found to be not only crucial for pellicle formation, but also heterogeneously expressed within the pellicle. The components of the extracellular polymeric substances (EPS) in pellicles were analysed by confocal microscopy using lectins, revealing distinct pellicle morphology on the air-facing and medium-facing sides, and spatially- and temporally-regulated generation of the EPS components poly-N-acetyl glucosamine and colanic acid. We discuss the difference between pellicles formed by E. coli K-12, pathogenic E. coli strains and other species, and the relationship between E. coli K-12 pellicles and solid surface-attached biofilms.
Assuntos
Adesinas Bacterianas/metabolismo , Escherichia coli K12/metabolismo , Adesinas Bacterianas/genética , Biofilmes , Escherichia coli K12/genética , Polissacarídeos/metabolismoRESUMO
Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.
RESUMO
Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.
Assuntos
Escherichia coli , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Hormônio do Crescimento Humano/biossíntese , Anticorpos de Cadeia Única/biossínteseRESUMO
For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics and are commonly seen in antibiotic-resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gram-negative bacteria. Accumulation of the fluorescent compound ethidium bromide (EtBr) was measured in Salmonella enterica serovar Typhimurium SL1344 (wild type) and efflux deficient (ΔacrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase, and did not correlate with acrAB transcription. EtBr accumulation in the ΔacrB strains was high in exponential phase but dropped sharply later in growth, with no significant difference from that in SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps but instead was due to decreased permeability of the envelope in stationary phase. Transcriptome sequencing (RNA-seq) identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. IMPORTANCE This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.