ABSTRACT
The movement of ammonium across biologic membranes is a fundamental process in all living organisms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus ...protein (Amt/Mep/Rh) family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter AmtB specifically binds 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol (POPG). Upon POPG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted, to our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.—Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB. FASEB J. 33, 1989–1999 (2019). www.fasebj.org
•Adsorption of PCR amplicons electrochemically detected on low-cost PCB electrodes with methlyene blue (MB).•Amplicon length and MB concentration are both critical to achieving linear sensor ...performance.•Detection of SARS-CoV-2 nucleocapsid gene amplicons from 10 pg/μl (1.7 fM).•N1 fragment successfully amplified and detected in wastewater spiked with SARS-CoV-2 control RNA.
We present a low-cost electrochemical DNA biosensor based on printed circuit board (PCB) electrodes for wastewater monitoring using portable PCR instruments, such as miniPCR®, without the requirement for qPCR reagents. PCB electrodes are attractive candidates for low-cost and sensitive DNA biosensors of relevance in a pandemic such as COVID-19, and facilitate the opportunity to map disease spread in Low-Middle Income Countries (LMICs) through monitoring of environmental samples such as wastewater. The biosensor reported in this work is capable of detecting PCR amplicons through the intercalation of methylene blue (MB) with DNA, which increases the voltammogram peak current at the redox potential of MB. We describe how these changes are likely to result from the adsorption of MB-DNA complex on the electrode surface. The electrodes are reusable, easy to clean, do not undergo any surface modification and represent a cost-effective solution with long shelf-life. We also explore the impact that MB concentration and DNA length have upon our biosensor performance and provide insights useful to other investigators in the field. The sensor reported here is capable of detecting SARS-CoV-2 nucleocapsid gene amplicons at concentrations as low as 10 pg/μl (approximately 1.7 fM) and can detect nucleotides amplified after 10 PCR cycles. Furthermore, using the PCB electrode and approaches described here, SARS-CoV-2 amplicons were detected in simulated wastewater sample, by spiking wastewater collected from a sewage treatment plant in Mumbai, India with SARS-CoV-2 RNA.
Plant growth-promoting endophytic (PGPE) actinomycetes have been known to enhance plant growth and mitigate plant from abiotic stresses via their PGP-traits. In this study, PGPE
Streptomyces
sp. GKU ...895 promoted growth and alleviated salt tolerance of salt-susceptible rice cultivar IR29 by augmentation of plant weight and declined ROS after irrigation with 150 mM NaCl in a pot experiment. Transcriptome analysis of IR29 exposed to the combination of strain GKU 895 and salinity demonstrated up and downregulated differentially expressed genes (DEGs) classified by gene ontology and plant reactome.
Streptomyces
sp. GKU 895 induced changes in expression of rice genes including transcription factors under salt treatment which involved in growth and development, photosynthesis, plant hormones, ROS scavenging, ion transport and homeostasis, and plant–microbe interactions regarding pathogenesis- and symbiosis-related proteins. Taken together, these data demonstrate that PGPE
Streptomyces
sp. GKU 895 colonized and enhanced growth of rice IR29 and triggered salt tolerance phenotype. Our findings suggest that utilisation of beneficial endophytes in the saline fields could allow for the use of such marginal soils for growing rice and possibly other crops.
Recent work has demonstrated that the polyketide natural product Aurodox from
is able to block the pathogenesis of the murine pathogen
In this work, we aimed to gain a better understanding of the ...mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagic
Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator,
Our data support a model in which Aurodox acts upstream of
and not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising antivirulence therapy for the treatment of these infections.
One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators (>8500 members in the Pfam database; Jan ...2009). These proteins contain a DNA-binding HTH domain at the N terminus of the protein and an effector-binding and/or oligomerisation domain at the C terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription. This review summarises what we know about the distribution, structure, function and classification of these regulators and suggests that they may have a future role in biotechnology.
Polymicrobial infection with
and
may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors, ...including metabolic processes and direct interaction of
with
hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, although the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of
/
whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form
in the biofilm founder population profoundly affects the structure of the biofilm as it matures. Different sub-populations of
and
arise within each biofilm as a result of the different
morphotypes, resulting in distinct sub-regions. These data reveal that
cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection.
Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A ...diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 - 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains.
C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10
substitutions per strain per year) than ST5 (5.6 × 10
substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains.
Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones.
The genus Streptomyces is widely recognized for its biotechnological potential. Due to a need to improve crops, clean up the environment and produce novel antimicrobial molecules exploiting ...Streptomyces has become a priority. To further explore the biotechnological potential of these organisms we analyzed the genome of the strain Streptomyces sp. Z38 isolated from contaminated roots tissues. Our analysis not only confirmed the ability of the strain to produce plant growth promoting traits but also a range of mechanisms to cope with the toxic effect of heavy metals through genes involved in metal homeostasis and oxidative stress response. The production of silver nanoparticles indicated that Streptomyces sp. Z38 may find utility in Green, Grey and Red biotechnology.
•Streptomyces is a bacterial genus with biotechnological potential.•Genome of the rhizospheric strain Streptomyces sp. Z38 was sequenced.•Traits of the strain include heavy metal resistance and plant-growth promoting.•Silver nanoparticles were produced by Streptomyces sp. Z38.
Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition ...element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing.
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