The Bacillus subtilis tyrZ gene encodes a highly selective tyrosyl-tRNA synthetase and is regulated by a MarR regulator and T box riboswitch

Abstract

Misincorporation of D-tyrosine (D-Tyr) into cellular proteins due to mischarging of tRNA(Tyr) with D-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding D-aminoacyl-tRNA deacylase, which prevents misincorporation of D-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when D-Tyr is present. We show that TyrZ is more selective for L-Tyr over D-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of D-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess D-Tyr is present.

Importance: Accurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds. Bacillus subtilis produces D-Tyr, an analog of L-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize a D-aminoacyl-tRNA deacylase to prevent misincorporation of D-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by which B. subtilis prevents misincorporation of D-Tyr in the absence of a functional D-aminoacyl-tRNA deacylase gene.

FIG 1

ywaE-tyrZ operon organization and mutations. The ywaE gene is depicted by a gray box, and the tyrZ gene is depicted by a black box. The T box riboswitch is depicted by a series of stem-loops between the ywaE and tyrZ genes, with the intrinsic transcriptional terminator labeled. The promoter is depicted by an arrow, and a terminator helix for the operon is depicted by a stem-loop. The locations of the spontaneous mutations in the ywaE gene used in the study (EΩ4 and E^opA7G) are indicated. The DNAs included in the lacZ reporter fusions are indicated below the operon. The location of the L101E mutation is indicated in the fusion for the ywaEL101E-tyrZ-lacZ fusion. All fusions were constructed in the absence (wild type) or presence of the A7G mutation.

FIG 2

Identification of a putative operator sequence. The promoter regions of ywaE genes found upstream of tyrZ genes in organisms related to B. subtilis were aligned. (A) Sequences were identified using BLASTp and aligned using the ClustalW algorithm. The putative −35 and −10 sequences, the SD sequence and start codons, and the predicted operator sequence are indicated. Bsub, Bacillus subtilis subsp. subtilis strain 168; BmojROH1, Bacillus mojavensis ROH1; BsSpizDV1, Bacillus subtilis subsp. spizizenii DV1; BspJS, Bacillus sp. strain JS; Blic14580, Bacillus licheniformis DSM 13; BspBT1B, Bacillus sp. strain BT1B; Batr1942, Bacillus atrophaeus 1942; BamyY2, Bacillus amyloliquefaciens Y2; areaBamyY, Bacillus amyloliquefaciens subsp. plantarum YAU B9601-Y2; Bsp5B6, Bacillus sp. strain 5B6. (B) A WebLogo of the predicted operator sequence was created using the WebLogo sequence generator program (42, 43). An arrow depicts the location of the A7G mutation identified.

FIG 3

Biofilm formation in the presence or absence of d-Tyr. Overnight cultures (5 μl) were spotted on modified MSgg plates in the presence or absence of d-Tyr (6 μM) and grown at 25°C for 3 days.