Physiological effects of anti-TRAP protein activity and tRNA(Trp) charging on trp operon expression in Bacillus subtilis

Abstract

The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to trp operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRNA(Trp). In this study, we examined the roles of AT synthesis and tRNA(Trp) charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRNA(Trp) level from 80% to 40%; the charged level decreased further, to 25%, in an AT-deficient mutant. Adding tryptophan with phenylalanine increased the charged tRNA(Trp) level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRNA(Trp) charging. Changes in the charged tRNA(Trp) level observed during growth with added phenylalanine were associated with increased transcription of the genes of tryptophan metabolism. Nutritional shift experiments, from a medium containing tryptophan to a medium with phenylalanine and tyrosine, showed that wild-type cultures gradually reduced their charged tRNA(Trp) level. When this shift was performed with an AT-deficient mutant, the charged tRNA(Trp) level decreased even further. Growth rates for wild-type and mutant strains deficient in AT or TRAP or that overproduce AT were compared in various media. A lack of TRAP or overproduction of AT resulted in phenylalanine being required for growth. These findings reveal the importance of AT in maintaining a balance between the synthesis of tryptophan versus the synthesis of phenylalanine, with the level of charged tRNA(Trp) acting as the crucial signal regulating AT production.

FIG. 1.

Signal molecules, regulatory proteins, and events influencing trp operon transcription in B. subtilis. The molecules presently known to participate in regulating the transcription of the trp operon are shown. The two signal molecules that play a regulatory role are Trp and tRNA^Trp. The two responding regulatory proteins are TRAP and AT. When Trp is plentiful, it activates the TRAP protein, which binds to trp operon leader RNA, promoting transcription termination. When cells are deficient in Trp, TRAP is inactive and transcription of the suboperon can proceed. When cells are deficient in charged tRNA^Trp, the AT protein is synthesized. AT binds to Trp-activated TRAP, reducing or preventing TRAP function. In addition, when there is a charged-tRNA^Trp deficiency, the synthesis of tryptophanyl-tRNA synthetase increases, improving the rate of tRNA^Trp charging.

FIG. 2.

Analysis of the tRNA^Trp charging levels in different strains grown under restricted conditions. Cultures of B. subtilis strains CYBS400 (wild type), CYBS223 (ΔmtrB), CYBS542 (↑AT), and CYBS318 (ΔrtpA) were grown in Vogel-Bonner minimal medium supplemented with 0.5% glucose and trace elements at 37°C. One hundred micrograms/ml Trp and/or 50 μg/ml Phe were added to selected cultures. Northern blot assays were performed using gel electrophoresis and a ³²P-labeled deoxyoligonucleotide complementary to the tRNA^Trp sequence (see Materials and Methods). The percent charged tRNA^Trp was calculated by dividing the amount of charged tRNA^Trp by the sum of the amounts of charged and uncharged tRNA^Trp. wt, wild type.

FIG. 3.

Analyses of the levels of charged and total tRNA^Trp following a growth shift from minimal medium plus Trp to minimal medium plus Phe and Tyr. (A) Cells of the wild-type strain (CYBS400) or the ΔrtpA mutant (CYBS318) grown to 120 Klett units in Vogel-Bonner minimal medium with 0.5% glucose, trace elements, and 100 μg/ml Trp at 37°C were harvested, washed with minimal medium, and then shifted to minimal medium with 0.5% glucose, trace elements, and 100 μg/ml Phe plus 100 μg/ml Tyr at 37°C. Following the shift, samples were taken at the times indicated and were assayed. Northern blot analyses were performed, and the percent charged tRNA^Trp was calculated as described in the legend to Fig. 2. The percent total tRNA^Trp was calculated by dividing the sum of the charged and uncharged tRNA^Trp bands for each lane relative to the level obtained in the sample from time 0. (B) Plot of the changes in the two cultures in percent charged tRNA^Trp over time. (C) Plot of changes in the percent total tRNA^Trp over time. wt, wild type.

FIG. 4.

Growth of various strains under specific culture conditions. Cultures of B. subtilis strains CYBS400 (wild type), CYBS223 (ΔmtrB), CYBS542 (↑AT), and CYBS318 (ΔrtpA) were grown in Vogel-Bonner minimal medium with the various supplements indicated plus 0.5% glucose and trace elements at 37°C. Cell density was determined hourly by using a Klett-Summerson colorimeter. Klett units were plotted versus time to obtain the growth curves shown. (A) Growth with or without 100 μg/ml Trp. (B) Growth with 50 μg/ml Phe with or without 100 μg/ml Trp. (C) The mtrB gene was deleted from the genome of the B. subtilis strain BS166 trpE26. Growth rates were determined for cultures of strains BS166 trpE26 and BS166 trpE26 ΔmtrB, as shown in panel A. Cultures were grown with the supplements indicated in the figure: 100 μg/ml Trp with or without 50 μg/ml Phe and 50 μg/ml Tyr.