Massive over-representation of solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family in the genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860.

Lineage-specific expansion (LSE) of protein families is a widespread phenomenon in many eukaryotic genomes, but is generally more limited in bacterial genomes. Here, we report the presence of 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family, within the 8.2 Mb genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860, a gene family over-representation of unprecedented abundance in prokaryotes. Representing over 6 % of the total number of coding sequences, the SBP genes are distributed across the whole genome but are found rarely in low-GC islands, where the gene density for this family is much lower. This observation, and the much higher sequence identity between the 434 Rhodoplanes TTT SBPs compared with the average identity between homologues from different species, is indicative of a key role for LSE in the expansion. The TTT SBP genes were found in the vicinity of genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds. A smaller expansion of TTT SBPs is known in some species of the β-proteobacteria Bordetella and we observed similar expansions in other β-proteobacterial lineages, including members of the genus Comamonas and the industrial biotechnology organism Cupriavidus necator, indicating that strong environmental selection can drive SBP duplication and specialisation from multiple evolutionary starting points

A microbubble-sparged yeast propagation–fermentation process for bioethanol production

Background Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation–fermentation bioethanol process was developed and analysed. Results Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. Conclusions The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation

Systems analyses reveal the resilience of Escherichia coli physiology during accumulation and export of the nonnative organic acid citramalate

© 2019 Webb et al. Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids. IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.We thank the Biotechnology and Biological Sciences Research Council UK and InnovateUK for funding (Industrial Biotechnology Catalyst BB/N01040X/1) and the Centre of Excellence in Mass Spectrometry funded by Science City York (Yorkshire Forward, EP/K039660/1, EP/M028127/1)

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets

Application of a Quantitative Carrier Test to Evaluate Microbicides against Mycobacteria

Abstract Microbicides for reprocessing heat-sensitive medical devices, such as flexible endoscopes, must be mycobactericidal to reduce the risk of nosocomial infections. Suspension test methods currently used for efficacy evaluation lack the stringency required for assessing inactivation of mycobacteria on surfaces. The quantitative carrier test method reported here is based on mycobacteria-contaminated reference carrier disks of brushed stainless steel. Each disk was contaminated with 10 L of a suspension of Mycobacterium terrae containing a soil load. Each disk with a dried inoculum was placed in a glass or Teflon vial, and then overlaid with 50 L of the test formulation or 50 L saline for the control carriers. Five test and 3 control disks were used in each run. At the end of the contact time, each vial received 9.95 mL neutralizer solution with 0.1% Tween-80 to stop the reaction and perform the initial microbicide dilution. The inoculum was eluted by mixing on a Vortex mixer for 60 s, and the eluates and saline used to subsequently wash the vials and the funnels were membrane-filtered. Filters were placed on plates of Middlebrook 7H11 agar and incubated at 37C for at least 30 days before colonies were counted and log10 reductions were calculated in colony-forming units. Tests with a range of commercially available products, having claims against mycobacteria, or believed to be broad-spectrum microbicides, showed that the method gave reproducible results. Products used included oxidizing agents (sodium hypochlorite and an iodophore), a phenolic, a quaternary ammonium compound, and ortho-phthalaldehyde. This method represents a much more realistic evaluation than the currently used quantitative suspension test method for the evaluation of mycobactericidal formulations for registration and, when performed at different product concentrations, allows an assessment of any safety margin or risks in using the test formulation in the field.</jats:p

Carrier Tests to Assess Microbicidal Activities of Chemical Disinfectants for Use on Medical Devices and Environmental Surfaces

Abstract For well over a decade, many deficiencies have been identified in current AOAC methods used to assess the microbicidal activities of chemical disinfectants on medical devices and environmental surfaces. This report discusses the development of quantitative carrier tests (QCT) designed to address these concerns. Decontamination of surfaces with dried inocula is invariably more difficult than when microorganisms are in suspension. For medical device as well as environmental decontamination, microbicides are used on contaminated surfaces, thus making it necessary to evaluate their microbicidal action on representative carrier materials contaminated with a dried challenge microorganism(s). Our approach is a 2-tiered QCT. The first tier (QCT-1) uses relatively ideal conditions to assess performance of the microbicide for screening purposes; the test uses smooth glass surfaces and quantities of disinfectant in excess of those likely to be experienced in the field. The second tier of testing (QCT-2) is more stringent because it uses (1) disks of brushed stainless steel as carriers, (2) only 50 μL of the test formulation on each carrier as compared to 1 mL in QCT-1, and (3) an added soil load to simulate the presence of residual body fluids or accumulated surface dirt. This review also discusses the factors that affect disinfection of medical devices and environmental surfaces in the context of the methodology used to evaluate the potency of microbicides. Specific recommendations for discussion are included, and performance criteria are suggested based on a risk-reduction approach for different classes of disinfectants. The focus is on improving the relevance of the test methodology to actual field use of disinfectants for devices and facilities in health care, and potentially in other settings. It is hoped that this review and its recommendations will initiate needed discussion and resolution of the many issues identified.</jats:p

Clospore: A Liquid Medium for Producing High Titers of Semi-purified Spores of Clostridium difficile

Abstract Clostridium difficile continues to cause infections in healthcare and other settings. Its spores survive well indoors and require sporicidal chemicals for infection control. However, proper testing of disinfectants is impeded due to difficulties in obtaining viable spores of high enough quality and titers to meet current regulations for sporicidal claims. A new liquid medium (Clospore) has been developed, based on a systematic review of the compositions of 20 other available media. C. difficile spores grown in the new medium and treated with a mixture of lysozyme and trypsin yielded final suspensions with &amp;gt;109 CFU/mL of viable spores, with a purity of &amp;gt;91 as tested by spore-staining and phase-contrast microscopy. The spores showed a biological decay rate of about 0.1 log10/month when dried on metal disks and stored indoors (air temperature 23 2C; relative humidity 52.76 15.08). Heating the purified spore suspensions to 70C for 10 min to inactivate any vegetative cells showed no spore activation or inactivation. The spores could be stored for at least 14 months either refrigerated (4C) or frozen (20 or 80C) in 50 (v/v) ethanol with virtually no loss in viability. The resistance of the enzyme-treated spores to three levels of sodium hypochlorite (1000, 3000, and 5000 ppm), using a standardized quantitative carrier test, was almost identical to that of the spores concentrated by centrifugation alone. The described procedure has been successfully applied to four standard (ATCC) and six clinical strains of C. difficile.</jats:p

Chemical disinfection of human rotavirus-contaminated inanimate surfaces

SUMMARYFomites may play a role in the transmission of rotavirus infections, and in view of this, 27 disinfectants were evaluated for their ability to inactivate human rotavirus (HRV) on contaminated non-porous inanimate surfaces. Disks of stainless steel, glass and two types of plastics were contaminated with about 107 plaque-forming units of HRV suspended in faecal matter. The inoculum was allowed todry and an equal volume of the product under test was applied to the contaminated surface. After contact for 1 min, the action of the disinfectant was stopped by dilution. Surviving infectious virus on the disks was determined by plaque assay in MA-104 cells. A product was considered to be effective if itcould reduce the virus titre by at least 3 log10. Only 33·3% (9/27) of the formulations tested proved to be effective. Further testing of the effective products, which included antiseptics, instrument soaks and hard-surface disinfectants, showed that all of them could, in fact, reduce the virus titre on contaminated surfaces by at least 6 log10. These findings show the relative resistance of HRV to a widerange of chemical disinfectants in common use, and also emphasize the need for a more thorough evaluation of the virucidal potential of formulations regularly employed in attempts to prevent and control outbreaks of rotaviral diarrhoea.</jats:p