Article Figures & Data
Figures
- FIG. 1.
A. Schematic diagram of the chromosomal organization of the pyrimidine biosynthetic (pyr) genes in M. smegmatis. The pyrRBC-orf3045-carAB-pyrF operon is shown at the left; the pyrD and pyrE genes are unlinked and located away from the pyr operon. MSMEG3045 denotes an open reading frame of unknown function. The bent arrow indicates the start of transcription of the operon. A portion of the promoter-leader region of the transcript shown below illustrates the predicted secondary fold of the PyrR-binding loop (BL; shaded) in the RNA, the Shine-Dalgarno sequence of a putative ribosome binding site (SD), and start codon (shaded AUG) for pyrR, the first gene of the operon. B. DNA sequence on the pyr operon promoter-leader region; the nontemplate strand is shown in capital letters. The sequence is numbered with the first base of the pyrR start codon as +1. The sequence specifying the PyrR-binding loop is enclosed in a box and shaded, except for a box designating the putative Shine-Dalgarno sequence. Putative −35 and −10 promoter sequences are shown in boldface. MSMEG3041 indicates an open reading frame of unknown function (annotated as a member of the thiopurine S-methyltransferase family) that is transcribed in an opposite direction to the pyr operon. Regions deleted from the binding loop in pyr′-lacZ fusion plasmids pFS20 and pFS22 are denoted by Δ1; those deleted in pyr′-lacZ fusion plasmids pFS21 and pFS23 are denoted by Δ2.
- FIG. 2.
Construction of pyr′-lacZ fusion plasmids for characterization of regulation. A. Schematic diagrams showing the set of pJEM13-derived translational fusions in which the pyr leader, including the PyrR-binding loop region, pyrR ribosome-binding site (RBS), and first three codons of pyrR were fused in frame via a five-codon linker (Gly-Thr-Lys-Leu-Ala) to lacZ at codon 13 (left), and the set of pJEM15-derived transcriptional fusions in which the same pyr segments were fused to 56 bp of the lacZ leader so that the lacZ ribosome-binding site lies at the normal distance from the lacZ open reading frame (right). B. Diagram showing the locations of PCR primers (F1, F2, F3, R1, and R2) and segments of M. smegmatis DNA that were inserted upstream of lacZ to generate the plasmids described in the text and in Tables 2 and 3. Numbering of the sequence is as in Fig. 1B. C. Proposed secondary structures of the pyr leader RNA regions in the pyr′-lacZ fusion plasmids from which segments of the PyrR-binding loop were deleted (Table 3). Arrows denote the site of deletion. SD denotes the Shine-Dalgarno sequence of the presumed pyr ribosome-binding site.
- FIG. 3.
Proposed mechanism of PyrR-mediated regulation of the M. smegmatis pyr operon by translational repression.
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Descriptiona Reference or source M. smegmatis strains mc2155 Parent strain; ATCC 700084 31 CF1 Derivative of mc2155; ΔpyrD::gm Gmr This work CF22 Derivative of mc2155; ΔpyrR This work CF24 Derivative of CF1; ΔpyrR ΔpyrD::gm Gmr This work E. coli strain DH5α recA1 endA1 gyrA96 thi-1 relA1 hsdR17(rK− mK +) supE44 φ80lacZΔM15 Δ(lacZYA-argF)U169 28 Plasmids pJEM13 E. coli-Mycobacterium translational lacZ reporter shuttle vector; Kmr 34 pJEM15 E. coli-Mycobacterium transcriptional lacZ reporter shuttle vector; Kmr 34 pZErO2.1 Cloning vector; Kmr Invitrogen p2NIL Cloning vector; Kmr 26 pGOAL19 Plasmid carrying lacZ, sacB, and hyg genes in a PacI cassette; Apr Hygr 26 pGMΩ1 Plasmid carrying the aacC1 omega interposon in a SmaI cassette; Gmr 30 pFS1 M. smegmatis pyrR in pZERO2.1; Kmr This work pFS3 M. smegmatis pyrD in pZERO2.1; Kmr This work pFS3Δ1 pyrD in-frame deletion in pZERO2.1; Kmr This work pFS3Δ2 pFS3Δ1 pyrD in-frame deletion with omega interposon; Kmr Gmr This work pFS4 pJEM13 with −488 to +9b of pyrR gene; Kmr This workc pFS5 pJEM13 with −233 to +9 of pyrR gene; Kmr This workc pFS6 pJEM13 with −102 to +9 of pyrR gene; Kmr This workc pFS8 pJEM15 with −488 to +9 of pyrR gene; Kmr This workc pFS9 pJEM15 with −233 to +9 of pyrR gene; Kmr This workc pFS10 pJEM15 with −102 to +9 of pyrR gene; Kmr This workc pFS11 pZErO2.1 with −488 to +9 of pyrR gene; Kmr This work pFS14 pFS1 derivative, pyrR in-frame deletion; Kmr This work pFS16 pFS11 derivative, binding loop deletion (BLΔ1); Kmr This work pFS18 pFS11 derivative, binding loop deletion (BLΔ2); Kmr This work pFS19 pyrR in-frame deletion cloned in p2NIL; Kmr This work pFS20 pJEM13 with −233 to +9 of pyrR gene, BLΔ1); Kmr This workd pFS21 pJEM13 with −233 to +9 of pyrR gene, BLΔ2); Kmr This worke pFS22 pJEM15 with −233 to +9 of pyrR gene, BLΔ1; Kmr This workd pFS23 pJEM15 with −233 to +9 of pyrR gene, BLΔ2; Kmr This worke pFS24 pFS19 with pGOAL19 PacI cassette; Kmr Hygr This work pFS28 pJEM15 with −488 to +579 of pyrR gene (includes intact pyrR); Kmr This workc pFS30 pJEM15 with MSMEG_3041 promoter; Kmr This workc pFS31 pJEM15 with Ag80 promoter; Kmr This workf pFS32 pJEM15 with groEL2 (hsp60) promoter; Kmr This workf - TABLE 2.
Repression of M. smegmatis pyrB (ATCase) by uracil and derepression by pyrimidine-limiting growth on uridine require the pyrR gene
Function Strain Strain genotype Plasmid genotype Chromosomally encoded ATCase Plasmid-encoded β-galactosidase Sp act (nmol/min/mg) Ratio Sp act (nmol/min/mg) Ratio Uracil NE or uridinea Uracil NEa or uridine Repression mc2155 Wild type NAb 7.7 ± 0.2 19 ± 1.6 2.5 − − − CF22 ΔpyrR NA 64 ± 2.7 66 ± 4.1 1.0 − − − CF22/pJEM15c ΔpyrR None 64.5 ± 4.6 64.5 ± 2.9 1.0 3.5 ± 0.4 4.2 ± 1.9 1.2 CF22/pFS28d ΔpyrR pyrR-pyrB′-lacZ 11 ± 0.8 20 ± 2.0 1.8 500 ± 27 540 ± 6.9 1.1 Derepression CF1 ΔpyrD NA 9 ± 1.4 130 ± 6.2 14 − − − CF24 ΔpyrD ΔpyrR NA 69 ± 1.9 150 ± 7.8 2.2 − − − CF24/pJEM15 ΔpyrD ΔpyrR None 60 ± 1.4 180 ± 15 3.0 1.3 ± 0.1 6.8 ± 0.5 5.2 CF24/pFS28 ΔpyrD ΔpyrR pyrR-pyrB′-lacZ 9.1 ± 0.6 130 ± 21 14 470 ± 19 6,000 ± 260 13 ↵ a NE, no effector (for strains mc2155 and CF22); uridine was used for derepression of strains CF1 and CF24.
↵ b NA, not applicable.
↵ c pJEM15 is the vector plasmid (promoterless lacZ).
↵ d pFS28 is derived from pJEM15 and contains the pyr promoter region, pyrR, and the 5′ end of pyrB fused transcriptionally to lacZ (see text for details).
- TABLE 3.
Regulation of pyr′-lacZ transcriptional and translational fusion plasmids by pyrimidines
Strain Relevant genotype Plasmid Promoter regiona β-Galactosidase Sp act (nmol/min/mg) Ratio Uracilb Uridineb Translational fusions CF1 ΔpyrD pFS4 −488 to +9 400 ± 20 7,900 ± 330 20 CF1 ΔpyrD pFS5 −233 to +9 68 ± 4.1 3,200 ± 160 47 CF1 ΔpyrD pFS6 −102 to +9 6.7 ± 1.2 280 ± 33 42 CF1 ΔpyrD pJEM13 NAc 1.1 ± 0.44 0.76 ± 0.27 0.7 CF24 ΔpyrD ΔpyrR pFS5 −233 to +9 780 ± 99 3,500 ± 440 4.5 CF1 ΔpyrD pFS20 −233 to +9, with BLΔ1d 380 ± 21 2,000 ± 180 5.3 CF1 ΔpyrD pFS21 −233 to +9, with BLΔ2e 1,300 ± 55 6,300 ± 430 4.8 CF24 ΔpyrD ΔpyrR pFS20 −233 to +9, with BLΔ1 360 ± 25 1,300 ± 41 3.6 CF24 ΔpyrD ΔpyrR pFS21 −233 to +9, with BLΔ2 1,300 ± 140 4,900 ± 550 3.8 Transcriptional fusions CF1 ΔpyrD pFS8 −488 to +9 6,300 ± 300 15,000 ± 1,100 2.4 CF1 ΔpyrD pFS9 −233 to +9 4,400 ± 180 8,800 ± 640 2.0 CF1 ΔpyrD pFS10 −102 to +9 550 ± 59 1,000 ± 190 1.8 CF1 ΔpyrD pJEM15 NA 2.3 ± 0.20 8.6 ± 1.6 3.7 CF24 ΔpyrD ΔpyrR pFS9 −233 to +9 1,000 ± 77 6,500 ± 650 6.5 CF1 ΔpyrD pFS22 −233 to +9, with BLΔ1 1,100 ± 15 6,900 ± 500 6.3 CF1 ΔpyrD pFS23 −233 to +9, with BLΔ2 1,800 ± 75 11,000 ± 1,300 6.1 CF24 ΔpyrD ΔpyrR pFS22 −233 to +9, with BLΔ1 870 ± 30 4,900 ± 330 5.6 CF24 ΔpyrD ΔpyrR pFS23 −233 to +9, with BLΔ2 1,600 ± 63 8,700 ± 890 5.4 - TABLE 4.
Expression of alternative lacZ transcriptional fusion plasmids under pyrimidine-limiting conditions
Strain Relevant genotype Plasmid Promoter β-Galactosidase Sp act (nmol/min/mg) Ratio Uracila Uridinea CF1 ΔpyrD pFS30 MSMEG3041 380 ± 14 1,100 ± 100 2.9 CF1 ΔpyrD pFS31 Ag85A (fbpA) 77 ± 6.6 120 ± 21 1.6 CF1 ΔpyrD pFS32 hsp60 (groEL2) 8,300 ± 420 25,000 ± 2,200 3.0 CF24 ΔpyrD ΔpyrR pFS30 MSMEG3041 450 ± 30 1,000 ± 170 2.2 CF24 ΔpyrD ΔpyrR pFS31 Ag85A (fbpA) 57 ± 3.5 77 ± 8.3 1.4 CF24 ΔpyrD ΔpyrR pFS32 hsp60 (groEL2) 7,500 ± 180 21,000 ± 1,700 2.8 ↵ a Concentrations in medium: uracil, 100 μg/ml; uridine, 200 μg/ml.
