Adaptive Laboratory Evolution for Multistress Tolerance, including Fermentability at High Glucose Concentrations in Thermotolerant Candida tropicalis

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

Diverse Functions of Plant Zinc-Induced Facilitator-like Transporter for Their Emerging Roles in Crop Trait Enhancement

The major facilitator superfamily (MFS) is a large and diverse group of secondary transporters found across all kingdoms of life. Zinc-induced facilitator-like (ZIFL) transporters are the MFS family members that function as exporters driven by the antiporter-dependent processes. The presence of multiple ZIFL transporters was shown in various plant species, as well as in bryophytes. However, only a few ZIFLs have been functionally characterized in plants, and their localization has been suggested to be either on tonoplast or at the plasma membrane. A subset of the plant ZIFLs were eventually characterized as transporters due to their specialized role in phytosiderophores efflux and auxin homeostasis, and they were also proven to impart tolerance to micronutrient deficiency. The emerging functions of ZIFL proteins highlight their role in addressing important traits in crop species. This review aims to provide insight into and discuss the importance of plant ZIFL in various tissue-specific functions. Furthermore, a spotlight is placed on their role in mobilizing essential micronutrients, including iron and zinc, from the rhizosphere to support plant survival. In conclusion, in this paper, we discuss the functional redundancy of ZIFL transporters to understand their roles in developing specific traits in crop.

Genome-wide identification and characterization of SPX-domain-containing protein gene family in Solanum lycopersicum

The SYG1, PHO81, and XPR1 (SPX) domain is named after the suppressor of yeast gpa1 (Syg1), yeast phosphatase (Pho81) and the human Xenotropic and Polytrophic Retrovirus receptor1 (XPR1). SPX-domain-containing proteins play pivotal roles in maintaining phosphate ions (Pi) homeostasis in plant. This study was to genome-wide identification and analysis of Solanum lycopersicum SPX-domain-containing protein gene family. The Solanum lycopersicum genome contains 19 SPX-domain-containing protein genes. These SPX-domain-containing protein genes were located in seven of the 12 chromosomes. According to the different conserved domains, the proteins encoded by those genes could be divided into four SPX-domain-containing protein families, which included SPX Family, SPX-ERD1/XPR1/SYG1(SPX-EXS) Family, SPX-Major Facilitator Superfamily (SPX-MFS) Family and SPX-Really Interesting New Gene (SPX-RING) Family. Phylogenetic analysis of SPX-domain-containing protein genes in Arabidopsis thaliana, Solanum tuberosum, Capsicum annuum and Solanum lycopersicum classified these genes into eight clades. Expression profiles derived from transcriptome (RNA-seq) data analysis showed 19 SPX-domain-containing protein genes displayed various expression patterns. SPX-domain-containing protein may play different roles in phosphate nutrition of Solanum lycopersicum different tissues and development stages. And, this study can provide the selection of candidate genes for functional research and genome editing in Solanum lycopersicum phosphate ions (Pi) nutrition.

Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains

Background: Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. Methods: In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates. Results: Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes. Conclusion: Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.

Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9

The function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis. All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.

Genome-wide identification and analyses of tomato (Solanum lycopersicum L.) high-affinity nitrate transporter 2 (NRT2) family genes and their responses to drought and salinity

High-affinity nitrate transporter 2 (NRT2) proteins have vital roles in nitrate (NO3-) uptake and translocation in plants. The gene families coding NRT2 proteins have been identified and functionally characterized in many plant species. However, no systematic identification of NRT2 family members have been reported in tomato (Solanum lycopersicum). There is also little known about their expression profiles under environmental stresses. Accordingly, the present study aimed to identify NRT2 gene family in the tomato genome; then, investigate them in detail through bioinformatics, physiological and expression analyses. As a result, four novel NRT2 genes were identified in the tomato genome, all of which contain the same domain belonging to the Major Facilitator Superfamily (PF07690). The co-expression network of SlNRT genes revealed that they were co-expressed with several other genes in many different molecular pathways including transport, photosynthesis, fatty acid metabolism and amino acid catabolism. Programming many crucial physiological and metabolic pathways, various numbers of phosphorylation sites were predicted in the NRT2 proteins.

Membrane Transporters of the Major Facilitator Superfamily Are Essential for Long-Term Maintenance of Phenotypic Tolerance to Multiple Antibiotics in E. coli

We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions.

Performance of Scale

<p>Cities are for people. As the scale of modern day cities continue to grow with overbearing homogeneity, the human body has become a disengaged entity among the repetitive monolithic forms. This city environment has come to lack the facilitation of personal, social, spatial and economic connections. Architecture can be seen to be the major facilitator in engaging and shaping urban space and subsequently the connection between people and space. This thesis responds to these static environmental conditions, questioning how ‘shifts in scale’ could influence the performance of space, and resultantly how performative space can create a connection between the body and the city. The thesis identifies a gap in performance research which considers ‘shifting scales’ as a non-human active agent. Active agents are assessed for their effects on the body (subject) and space (form), becoming critical to successive design evaluation and development. The notion of ‘Performance in Architecture’ is defined and grounded through acknowledging current modes of discourse in architectural theory, establishing the relationships between the interconnected theories of ‘subject performance’ and ‘performance of form’. These two theories explore the performative relationships between the body ‘subject’ and architecture ‘form’, setting up the research structure and subsequent conditions for the production of progressive design iterations. The framework for the performance design iterations engages two alternate scaled conditions (Act One & Act Two), both of which are explored through parametric based software in conjunction with physical modelling. This parametric based technology enables the comprehension and fabrication of complex forms, allowing the design process to move between the digital world and real world with ease. Engaging with this technology allows the scaled conditions to become specifically responsive to parameters defined by the architect. Each design iteration ‘shifts in scale’, focusing on a select combination of components, providing feedback on the productive application as to how ‘shifts in scale’ influence the performance of space. The design iterations systematically bring together parameters responding to: Scaled Volume, Intersection, Context, Traversal Movement and Access, Circulation, Speed, and Connection. The resultant design is evaluated for its performative success in allowing the body to shift and move between multiple scaled volumes and floor levels. The production and evaluation of these design iterations grounds the importance of ‘shifts in scale’ as an active agent that generates a connection between the body and city. The uniqueness of each space provides a set of diverse tenanting opportunities where the design strategy begins to address the expansion and densification of cities, re-enlivening and connecting ‘unused’ ‘left over’ space. The design strategy acts as a catalyst for dealing with complex architectural parameters while maintaining its sensitivity to the human scale.</p>

Performance of Scale

<p>Cities are for people. As the scale of modern day cities continue to grow with overbearing homogeneity, the human body has become a disengaged entity among the repetitive monolithic forms. This city environment has come to lack the facilitation of personal, social, spatial and economic connections. Architecture can be seen to be the major facilitator in engaging and shaping urban space and subsequently the connection between people and space. This thesis responds to these static environmental conditions, questioning how ‘shifts in scale’ could influence the performance of space, and resultantly how performative space can create a connection between the body and the city. The thesis identifies a gap in performance research which considers ‘shifting scales’ as a non-human active agent. Active agents are assessed for their effects on the body (subject) and space (form), becoming critical to successive design evaluation and development. The notion of ‘Performance in Architecture’ is defined and grounded through acknowledging current modes of discourse in architectural theory, establishing the relationships between the interconnected theories of ‘subject performance’ and ‘performance of form’. These two theories explore the performative relationships between the body ‘subject’ and architecture ‘form’, setting up the research structure and subsequent conditions for the production of progressive design iterations. The framework for the performance design iterations engages two alternate scaled conditions (Act One & Act Two), both of which are explored through parametric based software in conjunction with physical modelling. This parametric based technology enables the comprehension and fabrication of complex forms, allowing the design process to move between the digital world and real world with ease. Engaging with this technology allows the scaled conditions to become specifically responsive to parameters defined by the architect. Each design iteration ‘shifts in scale’, focusing on a select combination of components, providing feedback on the productive application as to how ‘shifts in scale’ influence the performance of space. The design iterations systematically bring together parameters responding to: Scaled Volume, Intersection, Context, Traversal Movement and Access, Circulation, Speed, and Connection. The resultant design is evaluated for its performative success in allowing the body to shift and move between multiple scaled volumes and floor levels. The production and evaluation of these design iterations grounds the importance of ‘shifts in scale’ as an active agent that generates a connection between the body and city. The uniqueness of each space provides a set of diverse tenanting opportunities where the design strategy begins to address the expansion and densification of cities, re-enlivening and connecting ‘unused’ ‘left over’ space. The design strategy acts as a catalyst for dealing with complex architectural parameters while maintaining its sensitivity to the human scale.</p>