Toxin/antitoxin (TA) systems perhaps enable cells to reduce their metabolism to weather environmental challenges although there is little evidence to support this hypothesis. Escherichia coli GhoT/GhoS is a TA system in which toxin GhoT expression is reduced by cleavage of its messenger RNA (mRNA) by antitoxin GhoS, and TA system MqsR/MqsA controls GhoT/GhoS through differential mRNA decay. However, the physiological role of GhoT has not been determined. We show here through transmission electron microscopy, confocal microscopy and fluorescent stains that GhoT reduces metabolism by damaging the membrane and that toxin MqsR (a 5'-GCU-specific endoribonuclease) causes membrane damage in a GhoT-dependent manner. This membrane damage results in reduced cellular levels of ATP and the disruption of proton motive force (PMF). Normally, GhoT is localized to the pole and does not cause cell lysis under physiological conditions. Introduction of an F38R substitution results in loss of GhoT toxicity, ghost cell production and membrane damage while retaining the pole localization. Also, deletion of ghoST or ghoT results in significantly greater initial growth in the presence of antimicrobials. Collectively, these results demonstrate that GhoT reduces metabolism by reducing ATP and PMF and that this reduction in metabolism is important for growth with various antimicrobials.
Sponge gourd is a popular vegetable grown throughout India. Tomato leaf curl New Delhi virus, the causal virus of tomato leaf curl disease, has recently been reported to be associated with sponge gourd, causing up to 100% crop loss under epidemic conditions. We have collected 30 genotypically diverse genotypes of sponge gourd from different parts of India, screened these for resistance under natural epiphytotic conditions, and then confirmed the results through challenge inoculation with a purified strain of the virus under insect-proof greenhouse conditions. The minimum vulnerability index was recorded in genotype DSG-6 (3.33), followed by DSG 7 (6.0) under the challenge (whitefly-populated) inoculation conditions. Two susceptible genotypes ('Pusa Sneha' and NSG-1-11), both possessing desirable fruit characters, were crossed with the two most promising resistant lines (DSG-6 and DSG-7) and the disease reaction of segregating and backcross generations studied through challenge inoculation with a purified strain of virus under insect-proof greenhouse conditions. A chi-square (v 2 ) test of frequency distribution based on the vulnerability index of the F 2 progenies of the two resistant 9 susceptible crosses revealed monogenic dominant Mendelian ratio 3(R):1(S) to be the best fit in all crosses. This monogenic dominant model was further confirmed by the 1(R):1(S) ratio found to be best fit for the test cross with the susceptible parent. These results reveal that resistance to Tomato leaf curl New Delhi virus associated with yellow mosaic disease of sponge gourd is controlled by a single dominant gene in the genetic background of the resistant parents (DSG-6 and DSG-7) and that these two lines can be effectively utilized for the development of highyielding and yellow mosaic disease-resistant varieties/ hybrids of sponge gourd. This is the first conclusive identification of a resistant source and the inheritance of resistance against Tomato leaf curl New Delhi virus in sponge gourd.
Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.
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