Search

Mechanical forces shape living matter from the macro- to the microscale as both eukaryotic and prokaryotic cells are force wielders and sensors. However, whereas such forces have been used to control mechanically dependent behaviors in mammalian cells, we lack the same level of understanding in bacteria. Surface adhesion, the initial stages of biofilm formation and surface biofouling, is a mechanically dependent process, which makes it an ideal target for mechano-control. In this study, we employed nanometer surface vibrations to mechanically stimulate bacteria and investigate their effect on adhesion. We discovered that vibrational stimulation at the nanoscale consistently reduces surface adhesion by altering cell membrane potential. Our findings identify a link between bacteria electrophysiology and surface adhesion and provide evidence that the nanometric mechanical "tickling" of bacteria can inhibit surface adhesion.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella , with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Professor Owen R.T. Thomas reports financial support was provided by UK Engineering and Physical Sciences Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alexander Osgerby reports financial support was provided by Biotechnology and Biological Sciences Research Council., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 p araBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development., Competing Interests: Author TSC was employed by the company Cobra Biologics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zulkifly, Selas Castiñeiras and Overton.)

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 β-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-1β and a potential Neisserial vaccine candidate (BamA ENm ). 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., Competing Interests: Declaration of Competing Interest The Authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

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., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Masoura, Milner, Overton, Gkatzionis and Lund.)

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 (Δ acrB ) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase, and did not correlate with acrAB transcription. EtBr accumulation in the Δ acrB 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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H 2 O 2 ), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H 2 O 2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H 2 O 2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H 2 O 2 /gluconic acid. The susceptibility of catalase and general stress response sigma factor mutants confirmed the synergy of the three stressors, which is enhanced at higher H 2 O 2 concentrations. By monitoring cellular phenotypic changes caused by model honey, we explained how this can be bactericidal even though the antimicrobial compounds which it contains are at non-inhibitory concentrations.

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.

Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells using flow cytometry. Bacterial cells were stained with SYTO TM 84 to easily separate cells from background cell debris. Ethidium bromide fluorescence was then measured within the SYTO TM 84 positive population to measure accumulation. In S. Typhimurium SL1344, ethidium bromide accumulation was low, however, in a number of efflux mutants, accumulation of ethidium bromide increased more than twofold, comparable to previous whole population analysis of accumulation. We demonstrate simultaneous measurement of ethidium bromide accumulation and GFP allowing quantification of gene expression or other facets of phenotype in single cells. In addition, we show here that this assay can be adapted for use with efflux inhibitors, with both Gram-negative and Gram-positive bacteria, and with other fluorescent substrates with different fluorescence spectra., (Copyright © 2019 Whittle, Legood, Alav, Dulyayangkul, Overton and Blair.)

Graphene oxide (GO) has been reported to possess antibacterial activity; therefore, its accumulation in the environment could affect microbial communities such as biofilms. The susceptibility of biofilms to antimicrobials is known to depend on the stage of biofilm maturity. The aim of this study was to investigate the effect of GO nano-particles on Pseudomonas putida KT2440 biofilm of variable age. FT-IR, UV-vis, and Raman spectroscopy confirmed the oxidation of graphene while XPS confirmed the high purity of the synthesised GO over 6 months. Biofilms varying in maturity (24, 48, and 72 h) were formed using a CDC reactor and were treated with GO (85 μg/mL or 8.5 μg/mL). The viability of P. putida was monitored by culture on media and the bacterial membrane integrity was assessed using flow cytometry. P. putida cells were observed using confocal microscopy and SEM. The results showed that GO significantly reduced the viability of 48-h biofilm and detached biofilm cells associated with membrane damage while the viability was not affected in 24- and 72-h biofilms and detached biofilm cells. The results showed that susceptibility of P. putida biofilm to GO varied according to age which may be due to changes in the physiological state of cells during maturation. Graphical abstract.

Chlorine-based solutions are commonly used to sanitize orange fruits prior to juice extraction. We used flow cytometry (FCM) to investigate the physiology of Escherichia coli following its subjection to chlorine-based solutions and alternative sanitizing agents (H 2 O 2 and organic acids). Green fluorescent protein (GFP)-generating E. coli K-12 were washed with 50-200 ppm available chlorine (AC), 1%-5% H 2 O 2 , 2%-4% citric acid, 4% acetic acid, or 4% lactic acid, after which they were added to 1.2 μm-filtered orange juice (OJ). Cell physiology was investigated with FCM during storage at 4°C, and culturability was determined using plate counting. Analysis of GFP fluorescence allowed estimation of intracellular pH (pH i ). FCM results demonstrated an inverse relationship between the concentration of AC or H 2 O 2 and cellular health in OJ. Higher concentrations of sanitizer also resulted in a significantly greater number of viable but nonculturable (VBNC) cells. Real-time FCM showed that supplementation of AC with 2% citric acid, but not with 100 ppm of Tween-80, led to a significant reduction in pH i of the cells incubated in OJ, and that the majority of the reduction in pH i occurred during the first 2 min of incubation in OJ. Organic acids were found to be more effective than both AC and H 2 O 2 in reducing the pH i , viability, and culturability of the cells in OJ. The results confirmed the hypothesis that consecutive subjection of E. coli to maximum legally permitted concentrations of sanitizers and OJ induces the VBNC state. Furthermore, we demonstrate successful application of FCM for monitoring the efficacy of washing procedures., Competing Interests: TWO and AA were paid speaker expenses by BD for speaking at BD Accuri users' events.

Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters.

The development of a simple pH-stat fed-batch fermentation strategy for the production of Magnetospirillum gryphiswaldense MSR-1 and magnetosomes (nanoscale magnetic organelles with biotechnological applications) is described. Flow cytometry was exploited as a powerful analytical tool for process development, enabling rapid monitoring of cell morphology, physiology and polyhydroxyalkanoate production. The pH-stat fed-batch growth strategy was developed by varying the concentrations of the carbon source (lactic acid) and the alternative electron acceptor (sodium nitrate) in the feed. Growth conditions were optimized on the basis of biomass concentration, cellular magnetism (indicative of magnetosome production), and intracellular iron concentration. The highest biomass concentration and cellular iron content achieved were an optical density at 565 nm of 15.5 (equivalent to 4.2 g DCW·L -1 ) and 33.1 mg iron·g -1 DCW, respectively. This study demonstrates the importance of analyzing bacterial physiology during fermentation development and will potentially aid the industrial production of magnetosomes, which can be used in a wide range of biotechnology and healthcare applications., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Orange juice (OJ) contains numerous compounds some of which are known to play key roles in growth and survival of bacteria. This study aimed to investigate the effects of natural or processing-induced variations in OJ composition on the physiology of Escherichia coli . OJ and model OJ (MOJ) samples containing various sugars, organic acids, amino acids, or ascorbic acid were inoculated with E. coli K-12 MG1655 in different growth phases. The culturability, viability, and physiology of the cells were investigated during storage using plate counting and flow cytometry. Generally, stationary-phase cells displayed the greatest survival in both MOJ and OJ. Increase in incubation temperature from 4 to 22.5ºC caused a significant decrease in both healthy and culturable cell populations. Supplementation of MOJ with ascorbic acid and amino acids increased both the viability and culturability of the cells. Similar trends were observed in amino acid-supplemented OJ, albeit at a slower rate. In contrast, variations in sugar or organic acid composition had negligible effects on the physiological status of the cells. In summary, natural variation in ascorbic acid or amino acid concentrations could potentially have an adverse effect on the microbiological safety of orange juice.

Background: The extraction of biopharmaceuticals from plasma and serum often employs overly complicated antiquated procedures that can inflict serious damage on especially prone protein targets and which afford low purification power and overall yields. This paper describes systematic development of a high-gradient magnetic fishing process for recovery of immunoglobulins from unclarified antiserum., Results: Non-porous superparamagnetic particles were transformed into hydrophobic-charge induction adsorbents and then used to recover immunoglobulins from rabbit antiserum feedstocks. Comprehensive characterisation tests conducted with variously diluted clarified antiserum on a magnetic rack revealed that immunoglobulin binding was rapid (equilibrium reached in <45 s), strong (K d  < 0.1 mg mL -1 ), of high capacity (Q max  = 214 mg g -1 ), and pH and ionic strength dependent. In a high-gradient magnetic fishing process conducted with the same adsorbent, and a conventional 'magnetic filter + recycle loop' arrangement, >72% of the immunoglobulin present in an unclarified antiserum feed was recovered in 0.5 h in >3-fold purified form., Conclusions: Fast magnetic particle based capture of antibodies from an unclarified high-titre feed has been demonstrated. Efficient product recovery from ultra-high titre bioprocess liquors by high-gradient magnetic fishing requires that improved magnetic adsorbents displaying high selectivity, ultra-high capacity and operational robustness are used with 'state-of-the-art' rotor-stator magnetic separators. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 β-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. β-lactamase was fused to the C-terminal of scFv and β-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for β-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

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.

Nano-emulsions (typically droplet diameter<1μm) are common in foods, and have been extensively reported to present antimicrobial activity, however, the mechanism is not well defined, and some studies reported no effect. A review of the literature was conducted and revealed strongly contradictory reports regarding the antimicrobial effect of nano-emulsions even in reference to similar microbial species and formulations. Following up, this study aimed to investigate the effect of nano-emulsions on four bacterial species (Staphylococcus epidermidis, Bacillus cereus, Lactobacillus acidophilus and five Escherichia coli strains) possessing different surface charge and hydrophobicity. Model oil-in-water (O/W) emulsions with different size of oil droplets were prepared with sunflower oil stabilised by polysorbate 80 (Tween80) emulsifier (hydrophilic), using high shear mixing followed by ultrasonication. The viability of bacteria was monitored by culture, membrane integrity was assessed with flow cytometric analysis with propidium iodide (PI) staining and fluorescence microscopy monitored the spatial distribution of cells within the O/W emulsions. The stability of the nano-O/W emulsions in the presence of bacteria was assessed by monitoring the droplet size [D (4, 3)] and creaming height. In contrast to other reports the survival and growth of bacteria was not affected by the size of the oil droplets, no damage to the bacterial membrane was evident with flow cytometry and emulsion stability was not affected by the presence of bacteria during 7days of storage. Furthermore, the antimicrobial activity of caprylic acid (CA) was compared between O/W coarse and nano-emulsions while varying the concentration of the hydrophilic surfactant Tween80. The activity of CA was similar in nano-emulsion and coarse emulsion; however, it was higher than in bulk oil and was reduced with increasing Tween80 concentration, suggesting that its efficacy is dictated by formulation rather than oil droplet size. The results demonstrated no enhanced antimicrobial activity due to nano-sized oil droplets and that conclusions on nano-emulsions should be taken with caution., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Magnetotactic bacteria (MTB) are a diverse group of bacteria that synthesise magnetosomes, magnetic membrane-bound nanoparticles that have a variety of diagnostic, clinical and biotechnological applications. We present the development of rapid methods using flow cytometry to characterize several aspects of the physiology of the commonly-used MTB Magnetospirillum gryphiswaldense MSR-1. Flow cytometry is an optical technique that rapidly measures characteristics of individual bacteria within a culture, thereby allowing determination of population heterogeneity and also permitting direct analysis of bacteria. Scatter measurements were used to measure and compare bacterial size, shape and morphology. Membrane permeability and polarization were measured using the dyes propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol to determine the viability and 'health' of bacteria. Dyes were also used to determine changes in concentration of intracellular free iron and polyhydroxylakanoate (PHA), a bacterial energy storage polymer. These tools were then used to characterize the responses of MTB to different O 2 concentrations and iron-sufficient or iron-limited growth. Rapid analysis of MTB physiology will allow development of bioprocesses for the production of magnetosomes, and will increase understanding of this fascinating and useful group of bacteria.

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.

Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24h post-inoculation, compared to filtered OJ samples containing particles between 0.22 μm and 11 μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hour incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4 °C, while the FCM viable count did not substantially decrease until 48 h, decreases in TVC were observed between 0 and 48 hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in monitoring bacterial physiology in foods, and potential effects of OJ clarification on bacterial physiology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We describe a novel method of Polyhydroxyalkanoate (PHA) extraction using dimethyl sulphoxide (DMSO) for use in screening studies. Compared to conventional chloroform extraction, the DMSO method was shown to release comparable quantities of PHA from Cupriavidus necator cells, with comparable properties as determined using Fourier transform infrared spectroscopy and differential scanning calorimetry., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Current treatment routes are not suitable to reduce and stabilise bacterial content in some dairy process streams such as separator and bactofuge desludges which currently present a major emission problem faced by dairy producers. In this study, a novel method for the processing of desludge was developed. The new method, elevated pressure sonication (EPS), uses a combination of low frequency ultrasound (20 kHz) and elevated CO 2 pressure (50 to 100 bar). Process conditions (pressure, sonicator power, processing time) were optimised for batch and continuous EPS processes to reduce viable numbers of aerobic and lactic acid bacteria in bactofuge desludge by ≥3-log fold. Coagulation of proteins present in the desludge also occurred, causing separation of solid (curd) and liquid (whey) fractions. The proposed process offers a 10-fold reduction in energy compared to high temperature short time (HTST) treatment of milk.

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.

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.

The production of recombinant proteins is crucial for both the development of new protein drugs and the structural determination of drug targets. As such, recombinant protein production has a major role in drug development. Bacterial hosts are commonly used for the production of recombinant proteins, accounting for approximately 30% of current biopharmaceuticals on the market. In this review, I introduce fundamental concepts in recombinant protein production in bacteria, from drug development to production scales. Recombinant protein production processes can often fail, but how can this failure be minimised to rapidly deliver maximum yields of high-quality protein and so accelerate drug discovery?, (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.

A novel technique for technical-scale continuous purification of proteins is presented. It is based on the combined use of functionalized magnetic nano-particles and an Aqueous Micellar Two-Phase System featuring the non-ionic surfactant, Eumulgin ES, which undergoes temperature induced phase separation at ∼25°C. In the first step, conducted below the transition temperature (i.e. 15°C), the magnetic sorbent particles are added into the single dispersed phase and bind the protein of interest. Next, on raising the temperature to 30°C the protein-laden magnetic particles partition strongly into the micelle-rich top phase of the micellar two-phase system that's formed. The magnetically susceptible top phase is then continuously separated from the micelle-poor phase in a flowthrough tailor-made magnetic extractor featuring a permanent magnet providing an upwardly acting magnetic force. This separation device was shown to be effective for continuous separation of a wide range of differently sized magnetic particle sorbents (i.e. from 2μm diameter to as small as 25nm) from a 10% (w/w) Eumulgin ES system; high separation efficiencies were recorded for the phase-forming surfactant (87 to >98%), and all magnetic sorbent particles tested (95-99.9%). Finally, protein purification by continuous magnetic extraction was demonstrated at 15L scale for the recovery of an antibody fragment, A33 Fab', from a crude extract of Escherichia coli periplasm. Nearly 70% of the A33 Fab' initially present in the extract at 15.6% of the total protein content was recovered in a 2-fold concentrated and highly purified (>98%) state. Further, the amounts of magnetic sorbent and phase-forming surfactant lost in the process were very small; thus recycling of both components into subsequent rounds of continuous magnetic extraction is highly feasible., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

The ability of biofilms to withstand chemical and physical extremes gives them the potential to be developed as robust biocatalysts. Critical to this issue is their capacity to withstand the physical environment within a bioreactor; in order to assess this capability knowledge of their surface properties and adhesive strength is required. Novel atomic force microscopy experiments conducted under growth conditions (30°C) were used to characterise Escherichia coli biofilms, which were generated by a recently developed spin-coating method onto a poly-l-lysine coated glass substrate. High-resolution topographical images were obtained throughout the course of biofilm development, quantifying the tip-cell interaction force during the 10 day maturation process. Strikingly, the adhesion force between the Si AFM tip and the biofilm surface increased from 0.8 nN to 40 nN within 3 days. This was most likely due to the production of extracellular polymer substance (EPS), over the maturation period, which was also observed by electron microscopy. At later stages of maturation, multiple retraction events were also identified corresponding to biofilm surface features thought to be EPS components. The spin coated biofilms were shown to have stronger surface adhesion than an equivalent conventionally grown biofilm on the same glass substrate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux., Methodology/principal Findings: Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide., Conclusions/significance: These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress.

Background: Neisseria gonorrhoeae can survive during oxygen starvation by reducing nitrite to nitrous oxide catalysed by the nitrite and nitric oxide reductases, AniA and NorB. The oxygen-sensing transcription factor, FNR, is essential for transcription activation at the aniA promoter, and full activation also requires the two-component regulatory system, NarQ-NarP, and the presence of nitrite. The only other gene known to be activated by the gonococcal FNR is ccp encoding a cytochrome c peroxidase, and no FNR-repressed genes have been reported in the gonococcus. In contrast, FNR acts as both an activator and repressor involved in the control of more than 100 operons in E. coli regulating major changes in the adaptation from aerobic to anaerobic conditions. In this study we have performed a microarray-led investigation of the FNR-mediated responses in N. gonorrhoeae to determine the physiological similarities and differences in the role of FNR in cellular regulation in this species., Results: Microarray experiments show that N. gonorrhoeae FNR controls a much smaller regulon than its E. coli counterpart; it activates transcription of aniA and thirteen other genes, and represses transcription of six genes that include dnrN and norB. Having previously shown that a single amino acid substitution is sufficient to enable the gonococcal FNR to complement an E. coli fnr mutation, we investigated whether the gonococcal NarQ-NarP can substitute for E. coli NarX-NarL or NarQ-NarP. A plasmid expressing gonococcal narQ-narP was unable to complement E. coli narQP or narXL mutants, and was insensitive to nitrate or nitrite. Mutations that progressively changed the periplasmic nitrate sensing region, the P box, of E. coli NarQ to the sequence of the corresponding region of gonococcal NarQ resulted in loss of transcription activation in response to the availability of either nitrate or nitrite. However, the previously reported ligand-insensitive ability of gonococcal NarQ, the "locked on" phenotype, to activate either E. coli NarL or NarP was confirmed., Conclusion: Despite the sequence similarities between transcription activators of E. coli and N. gonorrhoeae, these results emphasise the fundamental differences in transcription regulation between these two types of pathogenic bacteria.

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P(aniA) is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P(norB) is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP(+) and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P(norB) by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism.

The periplasmic nitrate reductase of Escherichia coli is important during anaerobic growth in low-nitrate environments. The nap operon encoding this nitrate reductase comprises seven genes including a gene, napF, that encodes a putative cytoplasmic iron-sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the E. coli cytoplasm. A bacterial two-hybrid protein-protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His(6)-tagged protein, exhibited spectral properties characteristic of an iron-sulphur protein. This protein was able to pull down NapA from soluble extracts of E. coli. A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm.

The transcription factor FNR, the regulator of fumarate and nitrate reduction, regulates major changes as Escherichia coli adapts from aerobic to anaerobic growth. In an anaerobic glycerol/trimethylamine N-oxide/fumarate medium, the fnr mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to reveal the response to oxygen, nitrate, and nitrite in the absence of glucose repression or artifacts because of variations in growth rate. Hence, many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved. The current microarray data confirmed 31 of the previously characterized FNR-regulated operons. Forty four operons not previously known to be included in the FNR regulon were activated by FNR, and a further 28 operons appeared to be repressed. For each of these operons, a match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 103, and possibly as many as 115, operons. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL, and a further 41 operons are repressed. The narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new defense mechanisms against reactive nitrogen species.

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c', similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c' was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c' protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c' gene was overexpressed in Escherichia coli and recombinant cytochrome c' was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c' is similar to previously characterized cytochrome c' proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.