Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

Reclassification of Chromobacterium violaceum ATCC 31532 and its quorum biosensor mutant CV026 to Chromobacterium subtsugae

The precipitous drop in the cost of genomic sequencing and the concomitant availability of computational methods for comparing genome-level data has made the accurate taxonomic placement of bacteria affordable and relatively rapid. Inaccurate taxonomic placement of bacteria has serious implications in clinical, environmental, and regulatory microbiology, but it can also adversely affect interpretation of research results. The quorum biosensor strain CV026 was derived from an isolate of Chromobacterium that was labeled as C. violaceum ATCC 31532, and is catalogued by the ATCC under that species name. Nearly 200 papers have been published that use CV026 as an indicator for quorum sensing activity in many Gram negative bacteria, but the inability of C. violaceum strains to complement the quorum sensing mutation in CV026 has called the taxonomic placement of the parent strain into question. We used molecular phylogeny and a large number of metabolic and phenotypic characters to demonstrate that Chromobacterium strain ATCC 31532 is a member of species Chromobacterium subtsugae.

Reclassification of Chromobacterium violaceum ATCC 31532 and its Quorum Biosensor Mutant CV026 to Chromobacterium subtsugae

The precipitous drop in the cost of genomic sequencing and the concomitant availability of computational methods for comparing genome-level data has made the accurate taxonomic placement of bacteria affordable and relatively rapid. Inaccurate taxonomic placement of bacteria has serious implications in clinical, environmental, and regulatory microbiology, but it can also adversely affect interpretation of research results. The quorum biosensor strain CV026 was derived from an isolate of Chromobacterium that was labeled as C. violaceum ATCC 31532, and is catalogued by the ATCC under that species name. Nearly 200 papers have been published that use CV026 as an indicator for quorum sensing activity in many Gram negative bacteria, but the inability of C. violaceum strains to complement the quorum sensing mutation in CV026 has called the taxonomic placement of the parent strain into question. We used molecular phylogeny and a large number of metabolic and phenotypic characters to demonstrate that Chromobacterium strain ATCC 31532 is a member of species Chromobacterium subtsugae.

Rapid Characterization of Quorum Sensing Inhibitory Molecules from Garcinia indica Choisy Seed Methanol Extract by GC-MS Analysis

Background: Bacteria communicate by producing small signaling molecules to maintain threshold cell density. Quorum sensing inhibition is an effective and most potent approach for the prevention of cell to cell communication. Methods: In the present study, the methanol extract of Garcinia indica was tested for anti-quorum sensing activity using Chromobacterium violaceum as a biosensor. The extract inhibited violacein production in biosensor strain without affecting the growth of the bacteria and this was corroborated by bioautography assay. Results: The bioactive fraction was analysed by the aid of GC-MS revealed that, the presence of coumarin (7-hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin) and eugenol (1,2-dimethoxy-4-(1-methoxy-1- propenyl) benzene) derivatives as anti-quorum sensing molecules. Conclusion: A detailed investigation is required to study the mechanism of action involved in control of quorum sensing signals and possible applications.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct E. coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications

Reclassification of Chromobacterium Violaceum ATCC 31532 and its Quorum Biosensor Mutant CV026 to Chromobacterium Subtsugae

The precipitous drop in the cost of genomic sequencing and the concomitant availability of computational methods for comparing genome-level data has made the accurate taxonomic placement of bacteria affordable and relatively rapid. Inaccurate taxonomic placement of bacteria has serious implications in clinical, environmental, and regulatory microbiology, but it can also adversely affect interpretation of research results. The quorum biosensor strain CV026 was derived from an isolate of Chromobacterium that was labeled as C. violaceum ATCC 31532, and is catalogued by the ATCC under that species. Nearly 200 papers have been published that use CV026 as an indicator for quorum sensing activity in many Gram negative bacteria, but the inability of C. violaceum strains to complement the quorum sensing mutation in CV026 has called the taxonomic placement of the parent strain into question. We used molecular phylogeny and a large number of metabolic and phenotypic characters to demonstrate that Chromobacterium strain ATCC 31532 is a member of species Chromobacterium subtsugae.

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation and migration. Erk is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response however, involves a Vegfr-dependent mechanism that initiates concomitant with the wound inflammatory response. This work thus reveals a highly dynamic sequence in regenerative angiogenesis that was not previously appreciated. Altogether, this study demonstrates the utility of a unique biosensor strain for analysing dynamic endothelial Erk-signalling events and validates a new resource for the study of vascular signalling in real-time.

Extracts of medical plants suppress the SOS response and reduce mutagenesis in E. coli

One of the promising directions in the fight against the emergence and spread of farm animal microbiota resistance factors is the development and search for feed additives that can inhibit the SOSresponse. SOS-response is one of the main mechanisms of the occurrence of mutations in bacteria. Plants used in traditional medicine can be a promising source of safe substances that reduce the SOS-response of bacteria. A screening of plants potentially containing substances with antiSOS activity was performed. During the initial screening, the E. coli MG 1655 pRecA-lux biosensor strain with ciprofloxacin as RecA inducer was used. Seven plants were identified whose extracts reduced the expression of the RecA operon. In further experiments on bacteria exposed to antibiotics, we identified four plants whose exstracts significantly reduced the mutagenesis rate of clinical E. coli strains: Austrian broom (Cytisus austriacus), greater celandine (Chelidonium majus), walnut (Juglans regia) and smooth sumac (Rhus glabra).

Substrate-mediated regulation of the arginine transporter ofToxoplasma gondii

ABSTRACTIntracellular parasites, such as the apicomplexanToxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection.TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) inT. gondii. Here, we show that the abundance ofTgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain ofT. gondii, we demonstrate that parasites vary the expression ofTgApiAT1 in different organs within their host, indicating that parasites are able to modulateTgApiAT1-dependent uptake of Arg as they encounter different nutrient environmentsin vivo. Finally, we show that Arg-dependent regulation ofTgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in theTgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

ABSTRACTAzotobacter vinelandiiis a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium,A. vinelandiihas evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies ofA. vinelandiiproducing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability ofA. vinelandiito recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter geneamtBthat resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green algaChlorella sorokinianaconfirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of theA. vinelandiiin coculture. The nature and quantities of nitrogen released by urease andamtBdisruptions were further compared to strains reported in previous efforts that altered thenifLAregulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.