Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines

EL!BVIER

FEh4S Microbiology

Letters 137 (1996) 13- 18

Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines Sa-You1 Ghim, Robert L. Switzer

*

Department of Biochemistry, University of Illinois, 600 South Mathews Aue., Urbana, IL 61801, USA Received 24

November 1995;revised 14 December 1995; accepted 17 December 1995

Abstract

The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role. A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described. The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance. Twelve such mutants were isolated and sequenced. All resulted from point mutations in the pyrR gene. Keywords: Bacillus subtilis; Mutations;

Pyrimidine

biosynthesis;

PyrR; Regulatory

1. Introduction In Bacillus subtilis the genes of pyrimidine nucleotide biosynthesis are organized into an operon, which is regulated by exogenous pyrimidines by transcriptional attenuation at three points in the promoter proximal end of the operon [l-3]. Turner et al. [3] have proposed that transcriptional attenuation is modulated by the protein product of the pyrR gene, which is believed to bind in a pyrimidine nucleotide-dependent fashion to a portion of an antiterminator structure in the pyr mRNA, thereby preventing anti-terminator formation and promoting formation of a downstream transcription terminator structure. Remarkably, PyrR has also been shown to

* Corresponding author. Tel.: + 1 (217) 333 3940; Fax: (217) 244 5858; E-mail: [email protected] 0378-1097/96/$15.00 0 1996 Federation PII SO378-1097(96)00012-2

of European

+

1

Microbiological

protein; Transcriptional

attenuation

possess uracil phosphoribosyltransferase activity, even though its deduced amino acid sequence is only very distantly related to those of other bacterial uracil phosphoribosyltransferases [4]. The relationship between the enzymatic activity of PyrR and its ability to function as an RNA-binding attenuation protein is not yet clear. A detailed study of structure-function relationships with PyrR would illuminate the nature of RNA recognition by this protein, the means by which RNA binding is modulated by pyrimidine nucleotides, and the mechanism of catalysis by this unusual phosphoribosyltransferase. As a first approach to such studies, we have developed a method for the facile selection of pyrR mutants that have defects in the regulation of the pyr operon. Twelve pyr regulatory mutants were isolated and shown by nucleotide sequencing to result from point mutations in the pyrR gene. Societies.

All rights reserved

2. Materials

and methods

2.1. Isolation and characterization

of’ Parr mutants

The selection used was a modification of the procedure first described by Paulus et al. L-51,who showed that many revertants of the uracil-sensitive B. subtilis strain JH861 (trpC2
All I2 of the pyrimidine-tolerant mutants from JH86 1 regained pyrimidine sensitivity upon expression of plasmid-borne pyrR gene. Mutants transformed with the pWPl8 vector [IO] remained pyrimidine-tolerant. 2.2. Aspartate

trunscarbamylasr

mssa)

Cell cultures (55 ml) were grown in Spizizen minimal medium [ 1 I] with and without 50 pg uracil and uridine per ml to a cell density corresponding to an optical density at 436 nm of 1.0 and were harvested by centrifugation, washed and resuspended in 5.5 ml of 10 mM Tris HCI/ 1 mM EDTA buffer (pH 8.0). After sonic disruption the cell extract was centrifuged at 16 000 X g for 10 min, and the supernatant was used for assays. Aspartate transcarbamylase activity was determined as described previously [ 12.131. Protein concentrations were determined with the Coomassie protein reagent (Pierce, Rockford, IL) using bovine serum albumin as the standard protein. 2.3. PCR ampl$cation

and DNA sequencing

PCR amplification and DNA sequencing were performed with a DNA Thermal Cycler (Perkin Elmer Cetus) as described by the manufacturer. PCR primers used for amplifying a 860-bp chromosomal DNA fragment that included the entire pyrR gene were PyrR-A 1 (5’-CGCGTTCCCGAGGATATGGC3’) and PyrR/Rev (5’-GACCTGCCGAATACTTTTTGG-3’). Primers for direct sequencing of the 5’ to 3’ (from the pyr promoter) strand of the PCR-amplified pyrR gene were PyrR/For (5’-CTAAAACCCCTCTATGCTCTG-3’) PyrR-S9 (5’-CACGAAATGATCGAACGC-3’) PyrR-S 10 (5’-GGCAAAACGCCTTGCGG-3’), PyrR-S 11 (S-GCAACGATGAACCGCTTG-3’) PyrR-S 12 (S-GCGCTTGTTGATGTAGGC-Y), and PyrR-S 13 (5’-CGGGAAAAACATCCCGAC-3’). Primers used for sequencing of the 3’ to 5’ strand of the PCR-amplified pyrR gene were PyrR-S-9R (5’-GCGTTCGATCATTTCGTG-3’) PyrR-A6R (5’-CAATGCGTTCCGCAAGGCG-3’) PyrR-S 11R (5’-CAAGCGGTTCATCGTTGC-3’), PyrR-S 12R (5’-GCCTACATCAACAAGCGC-3’), PyrR-S 13R (5’-GTCGGGATGTTTTTCCCG-3’), and PyrR-S 14R (5’-GCAACCTCTCTGGATTGCCC-3‘). All primers were labelled by [‘y- 32P]ATPs for direct sequencing of PCR-amplified products, and the se-

S.-Y. Ghim, R.L. Switzer/FEMS

Microbiology

quence of the pyrR mutant DNAs was determined for both strands [ 141 with the jinol’” Sequencing System (Promega, Madison, WI). 32P-Labelled DNA fragments were separated on 6% polyacrylamide35% formamide gels.

3. Results and discussion Table 1 describes 12 independently isolated pyrimidine-tolerant mutants derived from strain JH861. All 12 strains were converted to pyrimidine sensitivity by transformation with a plasmid bearing the pyrR gene, but not by the vector plasmid. Aspartate transcarbamylase assays demonstrated that all of the mutant strains were strongly derepressed in pyrB expression and were resistant to repression by exogenous pyrimidines. Sequencing the DNA encoding PyrR from each of these mutants confirmed that each mutant resulted from a single point mutation in the pyrR gene (Fig. 1). Three of the 12 mutations presumably act by preventing formation of significant amounts of the PyrR protein. BGHS and BGH18 were translation termination mutants occurring at codons near the N-terminus of PyrR. BGH17 resulted from a mutation in the ribosome binding site for pyrR. It is somewhat surprising that such a mutation in the

Table 1 Aspartate

transcarbamylase

Strain

activities of Badus Mutation

a RBS, putative ribosome

Parent strain G+U Gln- 11 + Stop Arg-14 + stop Glu-23 + Ala Thr-41 + Ile Asp- 106 + Tyr Asp- 106 + Tyr Asp- 106 + Tyr Arg- 126 + Ile Val-134 -+ Gly Thr-156 + Ile Thr-156 + Ile binding site.

15

ribosome binding site would reduce formation of PyrR to such a low level as to obliterate regulation. This may reflect the fact that the translation initiation codon for PyrR is a rare UUG codon, which would be expected to be unusually sensitive to changes in the ribosome binding site that reduce the affinity of the mRNA for the ribosome. The remaining nine mutations were missense mutations in the pyrR coding sequence (Fig. 1). One of these mutations was independently isolated three times (BGH4, 14, and 19). The biochemical basis for this mutation is readily rationalized. It results in the replacement of a highly conserved Asp residue in the PRPP binding domain of the protein with a Tyr residue (Fig. 2). Originally identified as of probable functional significance because of its invariant occurrence in the amino acid sequences of many phosphoribosyltransferases and PRPP synthetases [ 151, this residue is now known from x-ray crystallographic analysis of the structures of glutamine PRPP amidotransferase [ 161 and orotate phosphoribosyltransferase [17] to interact specifically with the ribofuranosyl ring of PRPP and nucleotides bound to the active site of these enzymes. Thus, the Asp-to-Tyr mutant of PyrR would almost certainly fail to bind the nucleotide substrate UMP and would render the protein unable to respond to the co-repressing metabolite.

subtilis pyrR mutants Specific activity of ATCase (nmol mgNo

JH86 1 BGHl7 in RBS a BGH5 BGHl8 BGH22 BGH12 BGW BGH14 BGH19 BGH3 BGHIS BGHll BGH20

Letters 137 (1996) 13-18

’ min- ’ )

pyrimidines

+ Uracil and uridine (50 pg ml-’ each)

Repression ratio

420 3210 3750 3540 3670 1010 620 780 740 2610 980 1760 2150

3010 3350 3500 3610 980 540 640 680 1820 700 1190 1430

1.1 1.1 1.0 1.0 1.0 1.1 1.2 1.1 1.4 1.4 1.5 1.5

S.-Y. Ghim. R.L. Switzer/

16

FEMS Microbiology

Letters 137 (1996) 13-18

phoribosyltransferase activity or the ability to act as RNA-binding attenuation proteins. We are developing procedures for the facile subcloning and overexpression of these and other mutant PyrR proteins, so that defects in their uracil phosphoribosyltransferase activity or ability to bind to pyrimidine nucleotides or pyr mRNA can be investigated directly. The three-dimensional structure of PyrR is also being determined by x-ray crystallographic analysis, so that a detailed structure-function analysis based on the regulation-deficient mutants readily isolated as described in this work will soon be possible. It may seem surprising that all of the mutants with defects in the regulation of repression of the pyr operon were in the pyrR gene and that none were

Five other unique mutations were identified (Fig. 1). Interestingly, these mutations were distributed throughout the length of the coding sequence. Too little is yet known about the structure of PyrR for us to suggest a biochemical basis for their defects in regulation of the pyr operon, but when the sequences of the four known members of the PyrR family are aligned and the locations of the mutations are viewed in light of the alignment (Fig. 2), it appears that they occur in or very near to conserved residues. Further interpretation is difficult, because two members of the family, the PyrR sequences from Haemophilus influenzae [ 181 and from a Synechocystis species [19], have been identified on the basis of their deduced amino acid sequences only, and it is not known whether they possess either uracil phos-

+l -10 -35 TTOACAGAGGGTTTCTTTTCTGAAATAATAAACGAAGCTG~TAGATTCTTT-CAGTCCAGAGAGGCTGAG~GGAT~CG~TAGA

54 (BGH17) r

CGGGATGCGTGTATAGGCGCGCACCTTGTCCTAAAACCCCTCTATGCTCTGGCAGGAGGGGTTTTTTCTTCTATAT~CT~144 T(BGH5) C(BGH22) T(BGHl8) pyrJz---> 4 4 4 ~~CACATTGAATCAAAAAGCTGTCATTCTCGACG~~AGGC~TT~GACGGGCGCTGACCAGGATTGCTCACG~TGATCG~CGC~T RIAHEM IERN ILDEQA IRRALT MNQKAV A stop stw T(BGH12)

RBE

4

AAAGGAATGAATAACTGCATTCTTGTCGGCATT~GA~~GAGGGATTTACCTGGC~CGCCTTGCGG~CGCATTG~CAGATTGAG LVGIKTRGI YLAKRLAERIEQIE KGMNNCI I GGAAATCCTGTTACAGTCGGTG~TTGATATTACTCTTTACAGAGATGATCTTTCT G N P V T V G E IDITLYRDDLSKKT

AAAAAAACAAGCAACGATGAACCGCTTGTAAAA SNDEPLVK

234 28

324 58

414 88

(BGH4,14,& 19) GGTGCAGATATTCCGGTAGATATTACAGATCAGATCATG GAD I P V D I TDQKVI T(BGH3)

LVDQVL

YTGRTVRAGM

504 118

Y G(BGHI5)

4 b GATGCGCTTGTTGATGTAGGCA~ACCTTCCTCCATTCAGCTTGCAG~GCTTGTGGACAGAGGACACCGGGAGCTGCCGATCCGAGCGGAT DALVDVGEPSSIQLAYLVDRGHRELPIRAD I G T(BGH11&20) 4 TATATCGGGAAAAACATCCCGA~TCAAAGTCTGAAAAGTTAT DEVDQNDLVAIY PrSKSEKVMVQL YIGKNI I

594 148

684 178

G-CGAATAA

696

EN

181

E

Fig. I. Sites of mutation in the pyrR mutants described in this study. The nucleotide sequence of the pyrR gene and its 5’ flanking region [3] is shown and is numbered from the start of transcription [2] as + I. The deduced amino acid sequence for the PyrR protein is shown below. The - 35 and - 10 consensus sequences for the py promoter and the translation initiation codon for PyrR are shown in boldface, Nucleotides that were mutated in each mutant, listed in parentheses, are underlined with the nucleotide found in the mutant above an arrow. Corresponding changes in the amino acid sequence of PyrR in the mutants appear below the amino acid sequence with the residue that was replaced underlined and the amino acid in the mutant protein shown below in boldface. Outlined letters indicate the putative ribosome binding site (REVS) for the pyrR gene.

S.-Y. Ghim, R.L. Switzer/FEMS

Microbiology Letters 137 (1996) 13-18

17

I(BGHl2. mlzi~~

B. subtilis B. caldolyticus H. influenzae Synechocystis

m-QKAVILD

jiE&,

BQA1maLm

/-l) 1-m

X--QXAVVND BQAIREhLl%5 INiSIBB--It-IIID IiDRFI&l’ISn ISEBIXekBQ sp.~QIIBILS PLBIRBlZTR LASQV-S

I-

~I~I~Q NPVTVOEI~ a B. subtilis B. caldolyticus EAElWWJIW3 ASVPVOBLlDI BV TQRRvmLse INLPSMELW H. influenzae Synechocystis sp.E%WQI~Q V?WPVQAI~ %LDY(BGH4,14,&19)

subtilis B. caldolyticus H. influenzae Synechocystis

4 l3MNNCILVO~ lc5aezm QIWCVLV@X lcmYLERR

B.

TLDDLVIV@z WI=1 DLSELVLL@Z Yl%%EVPmQ

KTSNDEPLVK GADIPVDI - RTDDWEPLVXQTtWPFP DQBDKMPWS ---1KTRTPA

QSSQYLNI

XTXIPLSL

I(BGH3)

GtBGHlS)

4

4

D&vDvqps

sxQ&l?kyLWma @maW?xm

D&WDL@SPA

R%XkVL-

DhTDFQMA

X~%VI~ W2ALNEWPB~QVER&TL~-~HP~ sp. PutitiveFXPP binding sites

ISRALB

xm

I(BGHllkZ0) B. subtilis

B. caldolyticus H. infl uenzae Synechocystis

4 YIE%NImX

SfZlWWJQLDEVEQl4DL~IY VmIDQwSIH

rvQlglwllr88R SmIVWELsE -R D&VQmTBX sp. Fv@3ZLm

-IL

BE!QVlWYLQD PEnRDTTXLI

ENE m

OK KG

Fig. 2. Alignment of the amino acid sequences of kncwn bacterial PyrR homologues. Outlined letters denote amino acid residues that are conserved in all four sequences. The boxed sequences indicate the putative PRPP binding region. Mutations in PyrR described in this work are shown with arrows. References for the PyrR sequences are as follows: B. subtilis [2,3], B. caldolyticus [20], H. influenzue [18], and Synechocystis sp. [19].

found in the DNA specifying the attenuator regions of the operon. We believe that this result is, in fact, exactly what should be expected. This is because the action of three tandem attenuators is required to fully repress the genes encoding the enzymes of UMP biosynthesis [ 1,3]. Thus, a mutation in a single attenuator region would probably not lead to total loss of regulation; loss of regulation would require two or three simultaneous mutations in the attenuators, which would surely be much more rare than a single mutation in the pyrR gene. Finally, it should be noted that the selection we have used to isolate PyrR mutants could be readily modified to screen for B. subtilis mutants with defects in either uracil or uridine uptake and conversion to UMP.

Acknowledgements

This investigation was supported by National Institutes of Health grant GM 47112. We thank Robert J. Turner for providing the plasmid pTS 185.

References

[II

Lu, Y., Turner, R.J. and Switzer, R.L. (1995) Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression. J. Bacterial. 177, 1315-1325. [21Quinn, C.L., Stephenson, B.T. and Switzer, R.L. (1991) Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. J. Biol. Chem. 266, 9113-9127. 131 Turner, R.J., Lu, Y. and Switzer, R.L. (1994) Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism. J. Bacterial. 177, 1315-1325. [41 Martinussen, J., Glaser, P., Andersen, P.S. and Saxild, H.H. (1995) Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis. J. Bacterial. 177, 271-274. El Paulus, T.J., McGarry, T.J., Shekelle, P.G., Rosenzweig, S. and Switzer, R.L. (1982) Coordinate synthesis of the enzymes of pyrimidine biosynthesis in Batik subtilis. J. Bacterial. 149, 775-778. [61Lu, Y. and Switzer, R.L. (University of Illinois). (1995) Unpublished data. 171Nygaard, P. (1993) Purine and pyrimidine salvage pathways. In: Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (Sonenshein,

I8

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[I I]

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[14]

[15]

S.-Y. Ghim. R.L. S~l~itzer/FEMS

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Microbiology

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