JOURNAL OF BACTERIOLOGY, JUlY 1990, p. 3804-3812 Vol. 172, No. 7 0021-9193/90/073804-09$02.00/0 Copyright C) 1990, American Society for Microbiology Repression and Catabolite Repression of the Lactose Operon of Staphylococcus aureus BABAK OSKOUIANt AND GEORGE C. STEWART* Department ofMicrobiology, The University of Kansas, Lawrence, Kansas 66045 Received 22 February 1990/Accepted 27 April 1990

The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.

The uptake of lactose by Staphylococcus aureus takes operon. The glucose effect seems to be mediated via the place via a phosphoenol pyruvate-dependent phosphotrans- action of a negative regulator (catabolite repressor) with a ferase system (reviewed in references 25 and 28). Lactose is target site overlapping that of the lac operator. phosphorylated upon entry into the cell by the joint action of two sugar-specific components, enzyme 11Lac (EIILaC) and MATERIALS AND METHODS enzyme I11Lac (ElIlLac). The intracellular phosphorylated lactose is then cleaved into glucose and galactose-6-phos- Bacterial strains, media, and reagents. The bacterial strains phate by phospho-fi-galactosidase (14). The galactose-6- and plasmid constructs used in this study are given in Tables phosphate generated is further metabolized via a tagatose- 1 and 2. L broth, prepared by the method of Miller (21), was phosphate pathway (2). used for E. coli cultivation. S. aureus and Bacillus subtilis The lactose (lac) genes of S. aureus have been shown to be cultures were grown either in L broth or in tryptic soy broth inducible with the addition of either lactose or galactose to or on tryptic soy agar (Scott Laboratories). Antibiotics were the culture medium. Galactose-6-phosphate was found to be used at the following concentrations: ampicillin, 100 pLg/ml; the actual intracellular inducer (22). Additionally, the lac chloramphenicol, 10 ,ug/ml; kanamycin, 20 p.g/ml; and tetra- genes are subject to catabolite repression (20). The signifi- cycline, 20 ,i.g/ml. cance of the glucose effect lies in the fact that cyclic AMP, Other materials were obtained from the following sources: which plays a critical role in catabolite repression in Esche- antibiotics, lysozyme, lysostaphin, o-nitrophenyl-,3-D-galac- richia coli, is not found, at least in physiologically significant topyranoside (ONPG), and ONPG-phosphate, Sigma Chem- in ical Co.; bacterial alkaline phosphatase, restriction endonu- concentrations, S. aureus (3; unpublished data). The cleases, Klenow fragment of DNA polymerase I, and T4 mechanism ofcatabolite repression in gram-positive bacteria DNA ligase, Promega Biotec and Pharmacia, Inc.; [a- is unknown. 32P]dATP, Dupont, NEN Research Products. DNA oligonu- Our laboratory has reported the cloning of the structural cleotides were purchased from Genetic Designs Inc., Hous- genes of the staphylococcal lac operon (5-7, 29). These ton, Tex. genes are arranged as a heptacistronic operon, according to Genetic techniques. (i) Electroporation. S. aureus cells data obtained by nucleotide sequence analysis. The organi- were grown in L broth to an A540 of 0.2. The cells were then zation of the operon is presented in Fig. 1. The terminal chilled on ice for 10 min and were harvested by centrifuga- three genes in the operon, lacFEG, encode EIITLaC, EIILaC, tion at 3,000 x g for 10 min at 4°C. The pellet was washed and phospho-p-galactosidase, respectively (5, 7). The func- two times with 10 ml of ice-cold 0.5 M sucrose and was tions of the lacA-D determinants are currently under inves- suspended in 1 ml of the same solution. The cells were kept tigation (29). We previously reported the cloning of the gene on ice for 20 min. The cells were then once again harvested encoding the repressor of the lac operon (lacR), which was as before and in 1 ml of M found to lie immediately upstream of the promoter-proximal suspended cold 0.5 sucrose. end Electroporation was carried out by using 200 to 400 ,ul of this of the lacA-G genes (24). cell suspension. The cells were mixed with 0.1 to 1 ,ug of In this report, the nucleotide and deduced amino acid plasmid DNA and placed in a Gene Pulser cuvette with a sequences of lacR are presented, as are experiments to 0.2-cm electrode define the repressor target site, the lac operator. In addition, gap (Bio-Rad Laboratories). The electro- the porator was a Bio-Rad Gene Pulser equipped with a pulse promoter of the operon has been identified through controller. The settings for S. aureus transformations were deletion and gene fusion analysis. We will also present data as follows: voltage, 2.5 kV; capacitor, 25 ,uF; and resistance, pertaining to the mechanism of catabolite repression of this 100 fl. After electroporation, the cells were immediately plated on antibiotic-containing medium. * Corresponding author. (ii) Conjugation. E. coli conjugations were done essentially t Present address: Scripps Clinic & Research Foundation, La as described by Miller (21). The donor strain was grown Jolla, CA 92037. without shaking for 2 h before mating. The recipient strain 3804 VOL. 172, 1990 REGULATION OF S. AUREUS lac 3805

kilobase 0 2 3 4 5 6 7 8 pairs r -r -F T T 7r T 7r I

I I I FtIII I I I I I I 11 I I U I I 1 1 11111 1 II Ir 11 I E C CPS C C EPvSpP A Sp EHp Hp H Pv PHpE S Hp

. - ? IacR lacA /acB lacC lacD lacF lacE lacG Protein Size 27 28.5 15.4 19.0 33.9 36.6 11.4 62.7 54.6 (kD)

FIG. 1. The lactose operon of S. aureus. Arrows indicate orientations of genes. Abbreviations used to denote restriction endonuclease recognition sites are as follows: A, AvaI; C, ClaI; E, EcoRl; H, HindIII; Hp, HpaI; P, PstI; Pv, PvuII; and S, Sall. kD, Kilodalton. was grown at 42°C for 1 h before mating. The donor and present in HB101 would have a copy of TnO000 inserted at recipient strains were then mixed at a 1:8 donor-to-recipient various sites on the plasmid (13). Physical mapping of these ratio, and the suspension was filtered through a filter (pore plasmids was then used to determine the location of each size, 0.45 pum; Millipore Corp.). The filter was incubated on TnJOOO insertion site. The plasmids with the desired inser- a tryptic soy agar plate for 2 h. The cells were then harvested tions were then sequenced by using a pair of 17-base primers in 1 ml of tryptic soy broth, and 200 ,ul was plated on complementary to the right and left ends of the transposon appropriate selective media. (18). The sequences of the primers are TCAATAAGT (iii) Plasmid copy number determinations. The method of TATACCAT (-y) and CAACGAATTATCTCCTT (5). Sheer-Abramowitz et al. (31) was used. The nucleotide sequence analysis was done by using either DNA sequencing. The dideoxy-chain termination method SEQAID (obtained from D. Roufa, Kansas State University) was used for nucleotide sequence determination (30). Both or GENEPRO (Riverside Scientific Enterprises). single- and double-stranded templates were used. These Site-directed mutagenesis. Specific mutations and deletions templates were prepared by three methods. were generated by using the method of Kunkel et al. (17). (i) Subclone sequencing. Plasmid pMK4 (33) and the repli- RNA isolation and hybridization. The minilysate method cative forms of coliphage M13mpl8 and M13mpl9 (37) were described by Holmes and Quigley (15) for DNA isolation used as vectors for the subcloning of staphylococcal DNA was modified for isolation of RNA from S. aureus in the restriction fragments. following manner: (i) the pH of STET buffer was adjusted to (ii) Deletion sequencing. To generate a series of sequential 6.8, and the pH of the sodium acetate used for the precipi- deletions, the procedure of Dale et al. was used (8). tation of nucleic acids was adjusted to 4.8; (ii) the RNase (iii) TnlOOO-primed sequencing. E. coli F+ strain MG1063 inhibitor vanadyl ribonucleoside complex (Sigma Chemical (12) harboring plasmid pBO649 (24) was used as the donor Co.) was added to a final concentration of 15 mM before cell strain in conjugation with HB101 (4). The transconjugant lysis; (iii) RNase-free DNase (Boehringer Mannheim Bio- cells were selected on tryptic soy agar plates containing 20 chemicals) at a concentration of 2 U/,Il of sample was used pug of tetracycline per ml (for selection for the mobilized to eliminate any copurified DNA. Twenty microliters of total pBO649) and 25 ,ug of streptomycin per ml (for selection cellular RNA (350 ,ug/ml) was suspended in 50 [lI of Tris- against MG1063). Plasmid pBO649 would only be trans- EDTA, 30 ,il of 20x SSC (1 x SSC is 0.15 M NaCl plus 0.015 ferred to HB101 if a transposition of TnJOOO from the F M sodium citrate), and 20 ,ul of 37% formaldehyde, and the plasmid to pBO649 followed by cointegrate formation and mixture was heated at 60°C for 15 min. A Hybri.Dot Mani- mobilization occurred. Therefore, each pBO649 molecule fold (Bethesda Research Laboratories) was then used to transfer serial dilutions of the RNA to a nitrocellulose membrane which had been prewet in 20x SSC. After the TABLE 1. Bacterial strains used transfer, the membrane was baked in a vacuum oven at 80°C Reference for 100 min. The membrane was prehybridized in 50% Strain Genotype or source deionized formamide, 5x Denhardt solution (19), 0.1% so- x E. coli dium dodecyl sulfate (SDS), and 5 SSPE (lx SSPE is 0.18 CJ236 dutI ung-J thi-I relAl (pCJ105 17 M NaCl, 10 mM NaPO4, pH 7.7, 1 mM EDTA). A 0.25-ml Cmr) volume of the prehybridization solution was used per 1 cm2 HB101 hsdS20 supE44 ara-14 galK2 4 of membrane along with 100 jig of fragmented salmon sperm lacYl proA2 rpsL20 xyl-5 mtl-l DNA per ml. The prehybridization was at 42°C for 1 to 2 h. recA13 For lac-specific RNA detection, 100 RI of denatured plasmid JM83 ara A(lac-pro) strA thi (+80d 37 lacIq lacZAM15) JMo10 A(lac-pro) thi strA endA sbcBJ5 37 TABLE 2. Plasmids used hsdR supE (F' traD36 proAB+ Insert Vector Relevant lacIq lacZAM15) Plasmid (size (reference) markers MC1000 araDJ39 A(araABC-leu)7679 32 [kb]) galU galK A(lac)X74 rpsL thi pBO649 EcoRI (3.1) pMH109 (24) lacR+ lacP+ MG1063 F+ recA56 thi 12 pBO649APstI EcoRI (3.1) pMH109 lacR lacP+ pBO860 HincII (0.4) pMLB1034 (32) lacP-lacZ fusion B. subtilis 168 trpC2 K. Bott pBO882 HincIl (0.4) pLC4 (26) lacP-xylE fusion pBO887 EcoRI-Sall (1.8) pMK3 (33) lacR+ S. aureus 8325-4 r- 16 pBO2000 HincII (0.4) pMK4 (33) lacP+ RN4220 pGS604 EcoRI (3.0) pMH109 lac'DFEG' 3806 OSKOUIAN AND STEWART J. BACTERIOL.

1 2 3 4 ORF 27 1- LacR LLaCA

E Sa DR Sa C Sa DD D A CSaP S H D A HHC i0 III11 I. II -1 I -1 I I U I -r- 0kb l 2

--*- ---4- -4-# -4--* .4 -4-10 *- -*--*

-*4-- -- * - - - - #-4- 4- -4-

I, 9 i

FIG. 3. Nucleotide-sequencing strategy. Symbols: .-, subclones generated for sequencing; *-+, deletion clone sequences;

FIG. 2. RNA dot blot analysis. Total cellular RNA was isolated from S. aureus RN4220 grown in the absence of inducing sugar (U), Nucleotide sequence analysis. The nucleotide sequence of in the presence of 1% galactose as inducer (I), and in the presence of lacR and the promoter region of the operon was determined. 0.5% galactose plus O.5% glucose (G). The RNA was blotted onto a The sequencing strategy and a partial physical map of the nitrocellulose membrane and hybridized to a radioactive probe region are depicted in Fig. 3. The DNA sequence is pre- containing lacFEG sequences. Lanes 1 through 4 contain 2.8, 1.4, sented in Fig. 4. The lacR open reading frame (ORF) begins 0.7, and 0.4 ,ug of RNA, respectively. at position 1117 with a TTG codon and ends at position 1810. This ORF is preceded by a strong potential ribosome-binding probe, labeled with 32P by nick translation (19), was added to site sequence (AAAGGAGG, positions 1039 to 1046). The the prehybridized filters along with 50% formamide-4x lacR gene product is a polypeptide of 251 amino acids with a Denhardt solution-200 ,ug of salmon sperm DNA per ml- calculated molecular weight of 28,534. The amino acid 0.1% SDS-5x SSPE-10% dextran sulfate. The hybridization sequence shows extensive homology to that of deoR (9, 34, was allowed to proceed for 14 to 16 h at 42°C. The filter was 35), the repressor of the deoxynucleoside operon of E. coli then washed twice with 2x SSPE and 0.1% SDS at room (Fig. 5). Their overall homology is 26%. One region of temperature and twice with 0.2x SSPE and 0.1% SDS at substantial homology lies between residues 24 and 43 of 65°C. The filter was then air dried and subjected to autora- DeoR, which form the helix-turn-helix motif of this DNA- diography. binding protein (10). The homologous site on LacR, residues Enzyme assays. ,B-Galactosidase assays were done by the 22 to 4i, is predicted to be a helix-turn-helix sequence, method of Miller (21). Phospho-p-galactosidase assays were according to calculations based upon parameters described performed as previously described (24). Catechol dioxygen- by Dodd and Egan (10). The recognition sequence for Sall ase assays were performed as previously described (26). corresponds to the terminal two codons of the lacR ORF. Cloning of lacR utilizing SalI resulted in active repressor repressor was a fusion of lacR to vector RESULTS (24). This actually sequences, resulting in a protein which contains all of the Transcriptional control of the operon. RN4220 cells were amino acids of LacR with the addition of six residues at the grown under three different conditions: in L broth (unin- carboxyl terminus. duced conditions), in L broth supplemented with 1% galac- An ORF capable of encoding a protein of 26 kilodaltons tose (induced conditions), and in L broth supplemented with precedes lacR and is oriented in the opposite direction. This 0.5% galactose plus 0.5% glucose (conditions of catabolite ORF extends from position 765 to 75 in the sequence. The repression). Total cellular RNA was isolated from each ORF is preceded by a reasonable promoter-like sequence, as culture and transferred onto a nitrocellulose membrane. The indicated in Fig. 4, but there is neither a clear-cut ribosome- immobilized RNA was then hybridized to a radiolabeled binding site sequence nor an initiation codon preceding the probe, pGS604, which contains the 3' end of lacD, all of lacF ORF. However, a 27-kilodalton protein was detected in and lacE, and the 5' end of lacG. The results of this maxicell analysis of clones bearing the EcoRI fragment hybridization are shown in Fig. 2. In the absence of induc- carrying this ORF sequence (24). tion, only low levels of lac-specific mRNA were detected. The promoter (lacP) for the structural genes of the lac Induction resulted in an increase in the steady-state levels of operon was found to be situated approximately 230 base lac mRNA. Conditions of catabolite repression yielded pairs (bp) downstream of lacR. This sequence, which is a greatly reduced levels of lac-specific transcripts, despite the good match to the consensus E. coli-B. subtilis vegetative presence of the inducing sugar. The presence of lac-specific promoter, has been shown to be the lac promoter by means mRNA thus correlates with the levels of expression of the of deletion and gene fusion analyses (see below). The site of operon as indicated by phospho-p-galactosidase assays (24). transposon TnS5S insertion in strain KUS74, which resulted Repression and catabolite repression of the staphylococcal in constitutive expression of the operon (6), is indicated. lac operon appear to operate at the transcriptional level. This site (between positions 1963 and 1964) is located in the VOL. 172, 1990 REGULATION OF S. AUREUS lac 3807

v v v v v v v v v v v v CTCCAAATTCCAAAACAGCAACTCCAAAATTAAAAG 36 ACATTTCAGTATAAAGAAATATCGCATAAAGAAAAACATACCCGTCAAATTGCAGAAAAA 1296 T F Q Y K E I S H K E K H T R Q I A E K 80 v v v v v v CATTTCCCTACCATTCGGGAAATGCTTTTTACATACTGATTACTCTGTCATTAATGATTT 96 AluI AluI v v v v v v Sau3A CGATTTATAGCTAAAAAAGCTGCATCATTAATTGAAGATGGGGATACTTTGTTTTTCGGA 1356 v v v v v v R F I A K K A A S L I E D G D T L F F G 100 TACAACGGAAACCATGTCGTCATGTATGACCAAAGTAGCGTCGCTATCATAAGGTGTTCG 156 v v v v v v DraI CCAGGAACAACAGTGGAACTATTAGCAGAAGAAGTCAATCATCATACGCTCACAATTATT 1416 v v v v v v P G T T V E L L A E E V N H H T L T I I 120 ATCTTTATTGATAATAATTAAATTGTCGCCTTTAAAATGTGATATTAATCCTGCGGCAGG 2216 v v v v v v RsaI RsaI ACGAATTGTTTGCCGGTGTATAAAATTTTGTTAGAAAAACAAACAGCACATTTTCGTGTC 1476 v v v v v v T N C L P V Y K I L L E K Q T A H F R V 140 TTGTACAACGAGTGATGAACCTAGTACAACAAGGGTGTCAGCATGTTCAATTTTATTTAA 2276 v v v v v v Sau3A TATTTAATTGGTGGTGAAATGCGCCATATTACAGAAGCATTTGTAGGTGAAATGGCGAAC 1536 v v v v v v Y L I G G E M R H I T E A F V G E M A N 160 TGCCCTTATGATGGTAGGTTGATCTAACATTTCACCGTATAATACGATGTCCGGTCGAAT 336 v v v v v v v v v v v v GCCATGTTGGAAAAACTAAGATTTAGCAAGATGTTCTTTAGTAGTAACGCAGTAAATAAA 1596 GGCACCACCACAATTATCACAATGTTTCAAAGTTCTATCAATAACATCTGACTTCGTATA 396 A M L E K L R F S K M F F S S N A V N K 180

HindI I v v v v v v v v v v v v GGTGCTGTGATGACATCTACATTAGATGAAGCCTATACGCAACAACTGGCACTAAGTAAT 1656 AGATTTATGACATACATTACAATAAAAACGATTTAACGTGCCATGTAATTCATCAACATG 4456 G A V M T S T L D E A Y T 0 Q L A L S N 200 Sau3A ClaI ClaI v v v v v v v v v v v v TTGACTTCCAGCGTCTGAGTGCAAACCATCGATATTTTGCGTGATGACACCTAAAGATTG 516 TCAATTGAAAAATACTTGTTAATCGATCATACGAAAGTTGGCAAAGAAGATTTTACATCA 1716 S I E K Y L L I D H T K V G K E D F T S 220 v v v v v v TTGATTACGTTCTAATTTTGCAATCCAATCATGAACGATATTGGGCATCGTATCGACAAA 576 PstI v v v v v v Sau3A TTTTGTCAGTTAAACGAATTGACTGCAGTGGTCATGGACTATGAAGATGAAGAAAAAGTA 1776 v v v v v v F C Q L N E L T A V V M D Y E D E E K V 240 TAGTAAGCGCTTATGGCAGAAATTGATAAAACCTTCAGGATCATCTTCTAAATAATCACG 636 HindII v v v v v v SalI GCTTAACAAGTATTCTGGCGAAAGCCCATCTTTTGAAATTTCATCAAATAAGCCACCATT 696 v v v v v v GAAACGATTAAAACATATATTGAAGTAGTCGACTAAATTAAAAGTATAACTGCATTGTTA 1836 v v v v v v E T I K T Y I E V V D < 251 GAACGGAAATCTGGAACGCCACTTGCGACAGATACACCAGCACCTGTAAAAAATGTAATA 756 v v v v v v v v v v v v AAATATGTATCTATATGCTCATAAGTGAATGTGAATTTGCATTCATTTATGGGCTATTTT 1896 816 H4indII v v v v v v v v v v v 876 TTATGCAAATTTATGTCAACGGGAACCTCGACAAAGTTTTACTACAATTTCAGACGAATA 1956

-10 v v v v v v DraI DraI CCTAATTAAAACAACACCACAATATGAATGTTCACTTATTGTTTGAAAATACGTCATAAA 2016 v v v v v v CGATTATCAAAAGCGCAGTTTAAACAGTTCTTTTTAAAAATGAATTATACATATAATCGA 936 Tn551 Dral -35 v v v v v v CAAACATATTATAAACATATTATGTTGATTTATTGTTTGTTTATGTTTATAATTTAAACG 2076 v v v v v v ACAGTTAAATTATATTAGTTTTTACGCAATTTTTATTATAGAATAAGCATGTATATTAAA 996 -35 -10

v v v v v v ATTGATGTCTAAACATAAAGGATAATGACCCATCGCCTAAATAAAGGAGGCAATTTTACA 1 056 rbs v v v v v v TAATCAAATTGAAAGCCTTTTCTCAAGATTTTCATAACCTACAAATAAATATGTCACATT 2136 v v v v v v TTGAATAAACATGAACGTTTGGACGAAATTGCTAAACTAGTGAATAAAAAGGGCACGATA 1116 v v v v v v M N K H E R L D E I A K L V N K K G T I 20 TTAAGAAAGGTATTTCAAAATTAAAGTAAAAAGGAGTCTTATTATGGCGATTATTATTGG 2196 lacR rbs M A I I I G 6 DraI AluI lacA v v v v v v v v v v v v AGAACGAATGAAATCGTCGAAGGTTTAAATGTGTCTGATATGACAGTTCGAAGAGATTTA 117 6 TTCAGATGAAGCTGGCAAACGATTAAAAGAAGTCATCAAATCATACTTATTAGACAATAA 225 6 R T N E I V E G L N V S D M T V R R D L 40 S D E A G K R L K E V I K S Y L L D N K 26 v v v v v v HindII HindII ATTGAATTGGAAAATAAAGGGATTTTAACGAAGATTCATGGTGGTGCACGCAGTAATTCA 1236 v v v v v v I E L E N K G I L T K I H G G A R S N S 60 ATATGATGTTGTTGACGTAACAGAAGGACAGGAAGTTGACTTTGTTGATGCAACTTTGGC 2316 Y D V V D V T E G Q E V D F V D A T L A 46 ClaI v v v v v v TGTAGCAAAAGATGTTCAAAGTCAAGAAGGTAACTTAGGTATTGTTATCGATG 2369 V A K D V Q S Q E G N L G I V I D 63

FIG. 4. Nucleotide sequence of the lacRA region of S. aureus. The nucleotide sequence as well as the deduced amino acid sequence of LacR are presented. Potential ribosome-binding sites are underlined. The site of insertion of TnS51 (6) is indicated. Putative promoter sequences are designated by arrowheads which indicate the transcriptional polarity. The 10-bp directly repeated sequence (*) and the 12-bp invertedly repeated sequence (#) of the lac promoter region are indicated.

region downstream of lacR and upstream of the promoter. reaction that did not contain any deoxynucleoside triphos- The sequence of the N-terminal encoding region of lacA, the phates) to remove the 3'-protruding ends of the digested first structural gene of the operon, is also presented (Fig. 4). DNA molecules. Because PstI digestion leaves a 4-base 3' Introduction of a frameshift mutation into lacR. The lacR overhang, removal of these bases then would result in a determinant was identified as encoding the repressor of the change in the reading frame of the presumptive protein that lac operon through complementation studies (24). To con- is encoded within that region. The removal of these bases firm that this gene product is indeed the repressor, a frame- was confirmed by sequence analysis, and the resultant shift mutation was introduced into the PstI site within the plasmid (pBO649APstI) was then introduced into RN4220 gene (24) (Fig. 4). Plasmid pBO649 was first digested with and phospho-p-galactosidase activity was measured. The PstI, which cleaves this plasmid only once at a site within results of the enzyme assays are shown in Fig. 6. As can be the lacR-coding sequence. Subsequently, the 3'-to-5' exonu- seen, the introduction of the frameshift mutation caused cleolytic activity of T4 DNA polymerase was used (in a premature termination of the lacR protein product. This 3808 OSKOUIAN AND STEWART J. BACTERIOL.

LacR LNKHERLDEIAKLVNKKGTIRTNEIVEGLNVSDMTVRRDLIELENKGILT 50 lacR DeoR METRREERIGQLLQELKRSDKLHLKDAAALLGVSEMT IRRDLNNHSAPWLL 52 { helix-turn-helix } EcoRI PatI EcoF,RI LacR KIHGGARSNSTFQYKEISHKEKHTRQIAEKRFIAKKAASLIEDGDTLFFG 100 pBO649' Sail DeoR GGYIVLEPRSASHYL-LS--DQKSRLVEEKRRAAKLAATLVEPDQTLFFD 99

LacR PGTTVELLAEEVNHH-TLTIITNCLPVYKILLEKQTAHFRVYLIGGEMRH 149 Leu Thr Ala Val Met + DeoR CGTTTPWIIEAIDNEIPFTAVCYSLNTFLAL--KEKPHCRAFLCGGEFHA 147 Val 19 amino acids Wild-type TTG ACT GCA GTG GTC ATG LacR ITEAFVGEMANAMLEKLRFSKMFFSSNAVNLGAVMTS-TLDEAYTQQLAL 198 AAC TGA CGT CAC CAG TAC DeoR SNAIFKPIDFQQTLNNFCPDIAFYSAAGVHVSKGATCFNLEELPVKHWAM 197

LacR SNSIEKYLLIDHTKVGKEDFTSFCQLNELTAVVMD-YEDEEKVETIKTYI 247 Leu Thr Trp Ser Trp + 7 amino acids DeoR SMAQKHVLVVDHSKFGKVRPARMGDLKRFDIVVSDCCPEDEYVKYAQTQR 247 Mutant TTG ACG TGG TCA TGG AAC TGC ACC AGT ACC LacR EVVD 251 DeoR IKLMY 252 8 FIG. 5. The homology between LacR and DeoR. The deduced amino acid sequences of the lacR and deoR genes are aligned. Positions of identity (:) and conservative changes (-) are shown. Sixty-six positions of identity were obtained, giving an overall (U(a homology of 26%. (DCm 'a CL 4 _ 0 cl)t termination, along with the addition of seven different amino acids (because of the shift in reading frame), resulted in a loss of repressor activity. Note that pBO649APst still con- tains the sequences downstream of the lacR region that titrate the repressor (24). In wild-type pBO649, intact repres- sor displayed dominance over these sequences (i.e., the None Wild-type Mutant noninducible phenotype), but introduction of the frameshift FIG. 6. Generation of a lacR frameshift mutation. Plasmid causes the titrating regions to confer a lac-constitutive pBO649 carrying a 3.1-kilobase EcoRI fragment containing lacR was phenotype upon the cells. DNA sequence analysis has digested with PstI. The linearized plasmid was then made blunt confirmed that the PstI site is contained within the lacR- ended with the exonuclease activity of T4 DNA polymerase, and the coding region. These studies confirm that lacR is indeed the plasmid was recircularized by ligation. The mutation was confirmed regulatory gene of the staphylococcal lac operon. by sequence analysis. The mutant and wild-type plasmids were Fusion of lacP to lacZ. A introduced into S. aureus RN4220, and phospho-,3-galactosidase 355-bp HindIl fragment (positions assays were performed. Enzyme activity is expressed as nanomoles 1915 to 2270) containing the putative promoter of the operon of o-nitrophenol released per minute per milligram (dry weight) of plus the first 30 codons of lacA was cloned in frame to create cells. The cultures examined contained no plasmid (none), pBO649 a gene fusion to codon 9 of lacZ in plasmid pMLB1034 (32). (wild-type), or pBO649APstI (mutant). Assays were done with cells The resulting plasmid, pBO860, when introduced into E. coli grown in the absence of induction (1) and with cells grown in the MC1000 (32), directs ,-galactosidase activity by utilizing the presence of 1% galactose to induce the lac operon (U). lac promoter and the lacA ribosome-binding site. However, the expression of lacZ could not be repressed by the codon, in fact, P-galactosidase activity was actually reduced introduction of a lacR-bearing plasmid (pBO649) into the in the mutant (unpublished data). MC1000 (pBO860) cells. On the basis of maxicell analysis Operon fusions with xylE. To determine whether repres- (24) and lacR-lacZ fusion analysis (unpublished data), the sion could be demonstrated in a gram-positive background, LacR protein does appear to be synthesized in E. coli. The lacP was cloned into the shuttle vector pLC4 (26), which lacR ORF could potentially be extended 11 codons upstream carries the promoterless reporter gene xylE. The lacP HindII of the initiator TTG codon, creating an ORF which initiates fragment was subcloned from pBO860 as an EcoRI-BamHI with an ATG. Initiation at this ATG may create a larger insert. This plasmid was designated pBO882. When trans- LacR polypeptide lacking activity. However, there is no formed into competent B. subtilis 168 trpC2 cells, the discernible ribosome-binding site sequence associated with plasmid directed the synthesis of catechol dioxygenase, as this putative ATG initiation codon and, therefore, it is not determined by the generation of a yellow color when the likely to be significantly utilized (1). Because the initiation colonies were sprayed with 0.5 M catechol (26). These cells codon for lacR is TTG (Fig. 4), the level of LacR in these were then made competent and transformed with a second cells may have been too low to allow for significant repres- plasmid, pBO887. This plasmid carries lacR as an EcoRI- sion of the transcription. TTG is rarely found as an initiation SalJ fragment in the shuttle vector pMK3 (33) and is com- codon in E. coli, and it appears to be inefficiently utilized as patible with pBO882. Cells resistant to both chloramphenicol well (27). Site-directed mutagenesis was used to change the (pBO882) and kanamycin (pBO887) were selected, and cat- TTG initiation codon to an ATG codon. The modified lacR echol dioxygenase assays were performed (Fig. 7). With gene was still not able to effect repression of lacP in E. coli these cells, expression of the xylE signal gene was repressed cells. Expression of P-galactosidase from lacR-lacZ fusions in trans by the presence of lacR. There was approximately a representing both the wild-type (TTG) and mutant (ATG) 25-fold reduction in xylE activity when pBO887 was present initiation codons indicated that translational efficiency in E. in the cells. The addition of galactose-6-phosphate to the coli was not improved with the introduction of the ATG culture did not result in the induction of lac-promoted xylE VOL. 172, 1990 REGULATION OF S. AUREUS lac 3809

TABLE 3. Titration of regulatory proteins of the S. aureus lac operon

Plasmida Sugarb Spp-galactosidasecact of phospho- pMK4 None 1.5 Glu + Gal 0.4 pBO2000 None 6.3 Glu + Gal 4.2 pA-60 None 2.7 Glu + Gal 0.6 pA-80 None 3.7 Glu + Gal 2.1 pA-60 A-80 None 1.4 0 Glu + Gal 0.7 200 pA-20 to -80 None 1.0 Glu + Gal 0.9 O 100 a Host strain was S. aureus RN4220. b L-broth cultures. Conditions of catabolite repression = 0.5% glucose 0 (Glu) plus 0.5% galactose (Gal). c Nanomoles of o-nitrophenol per minute per milligram (dry weight) of pBO882 + pBO887 pBO882 cells. FIG. 7. The lacP-xylE operon fusion. Plasmid pBO882 was con- structed such that the xylE gene of pLC4 (26) is under the control of lacP. The catechol dioxygenase activity encoded by this plasmid in presence ofglucose, despite both plasmids carrying the same a B. subtilis 168 trpC2 host was determined. The cells carried staphylococcal DNA insert (Table 4). pBO882 together with pBO887 (pMK3 carrying lacR) or pBO882 When the lacP insert was cloned upstream of the promot- together with the pMK3 vector (designated simply pBO882). En- erless xylE reporter gene (pBO882) and this construct was zyme activity is expressed as nanomoles of 2-hydroxymuconic introduced into S. aureus, expression of catechol dioxygen- semialdehyde formed per milligram (dry weight) of cells per hour. ase was reduced by 75% with the addition of glucose to the culture medium. The failure to repress expression more expression (data not shown). However, B. subtilis does not completely was likely due to the multicopy nature of the possess a galactose phosphotransferase system. It was not plasmid and a limited amount of the catabolite repressor. determined whether the galactose-6-phosphate entered the Expres'sion of xylE from pBO882 was not glucose sensitive cells in appreciable quantities. Therefore, the question of in B. subtilis hosts. induction of the lac operon in this heterologous host remains Generation of site-specific deletions in the lac promoter open. region. Within lacP there is a 10-bp perfect direct repeat, Catabolite repression of the lac operon in S. aureus. The with one half-site centered around -80 (relative to the start plasmid pBO2000 contains the 355-bp HindII lacP fragment site of transcription) and the other half-site centered at -28 cloned into the vector pMK4 (33). Introduction of this (44 bp of intervening sequence). There is additionally a 12-bp multicopy plasmid into S. aureus was found to influence the perfect inverted repeat situated with one half-site centered regulation of the chromosomal copy of the lac operon. The around -60 and the other centered at -24 (26 bp of presence of this plasmid confers constitutive expression of intervening sequence). The downstream half-sites of the the lac operon, as evidenced by significant expression of the directly and invertedly repeated sequences overlap by 6 bp operon in the absence of inducing sugar (Table 3). This effect and are positioned between the -35 and Pribnow box presumably results from the binding of the limited number of regions of the lac promoter. To ascertain any role played by the Lac repressor molecules by sequences carried by the these repeat sequences in regulation of the operon, the plasmid (24). Additionally, the lac region carried on the upstream half-sites were deleted by means of oligonucleo- plasmid interfered with catabolite repression of the operon tide mutagenesis. The mutagenic primers were 5'-GTTTA by glucose. In the presence of the plasmid, only approxi- mately 33% of the phospho-,-galactosidase activity is re- pressible by glucose addition. The pMK4 vector without the TABLE 4. Effect of plasmids on the interference lacP insert had no effect on catabolite repression. One of catabolite repressiona plausible mechanism to explain these observations is that the Plasmid Phospho-o-galactosidase lacP region on high-copy-number plasmids titrates a nega- activityb Copy number tive the operon which is activated by the regulator of None 0.1 presence of glucose in the medium. Consistent with this is pBO882 0.6 7 the observation that when the lacP insert is cloned into pBO2000 4.0 22 vectors with lower copy numbers, the inhibitory effect on catabolite repression of the chromosomal lac operon is a S. aureus RN4220 cells bearing the indicated plasmid were grown in L broth supplemented with 0.5% glucose plus 0.5% galactose (conditions of diminished. A threefold reduction in plasmid copy number catabolite repression). resulted in only 15% of the level of expression of the bNanomoles of o-nitrophenol released per minute per milligram (dry chromosomally encoded phospho-p-galactosidase in the weight) of cells. 3810 OSKOUIAN AND STEWART J. BACTERIOL.

TTATTGTTTGAAAATACGTCATAAACAAACATATTATAAACATATTATGTTGATTTATTGTTTGT-35

A-80 A-60 {l } A-20 to A-80 -10 TTATGTTTATAATTTAAACG

FIG. 8. Deletion analysis of lacP. The nucleotide sequence of the lac promoter region, as well as those sequences which were subjected to deletion analysis, are shown. The extents of the 9-bp deletions are indicated. The sequences are contained within the Hindll fragment and cloned in pMK4 (pBO2000 has the wild-type sequence).

TAATATGGACGTATTTTC-3' (designated A-60) and 5'- translational signals, the fusion protein was expressed in E. GACGTATTTTCGTGAACATTCAT-3' (A-80). The dele- coli hosts. Thus, the failure to detect repressor activity was tion mutants obtained, both singly and in combination, carry not likely due to failure of the E. coli cells to produce the a 9-bp deletion of the corresponding repeat half-site. The repressor. Changing of the TTG initiation to a more efficient deleted lacP sequences were subcloned into pMK4 and ATG codon (27) did not result in functional repressor activ- introduced into S. aureus, and their effect on lac regulation ity being detected in E. coli. Possible explanations are that was examined (Fig. 8 and Table 3). Catabolite repression an additional factor, present in gram-positive cells but not in was normal in strains bearing the plasmid with the -60 E. coli cells, is necessary for proper repressor function; that deletion. Therefore, this deletion appears to either eliminate the nature of repressor function is incompatible with the E. sequences involved in the binding of the catabolite repressor coli RNA polymerase; or that E. coli RNA polymerase can or disrupt critical spacing, and thus it cannot compete with utilize as a promoter a different sequence which is not chromosomal sequences for binding of this regulator. The affected by repressor binding. Further studies are required to -60 deletion also reduced binding of the LacR protein, address this problem. resulting in poorer competition with the chromosomal lac The promoter sequence identified for the structural genes operator site for binding. This was made evident by only ofthe staphylococcal lac operon resembles the consensus E. 43% of the phospho-p-galactosidase activity being displayed coli-B. subtilis vegetative promoter sequence. Identification in the absence of induction by the cells harboring the deleted of this sequence as the results from plasmid relative to cells with the wild-type sequence. Dele- promoter fusing this tion of the -80 repeat reduced binding of the catabolite sequence to a promoterless xylE signal gene and demonstrat- repressor by about 50% and had a lesser effect on binding of ing that production of catechol dioxygenase became lactose the LacR protein than did the -60 sequence deletion (59% of inducible and catabolite repressible in gram-positive cells phospho-i-galactosidase expression relative to the wild- bearing the fusion plasmid. In deletion studies, deletions type-plasmid-bearing cells). The combination of both dele- extending up to the identified Pribnow box or -35 regions tions destroyed the binding sites for both LacR and the did not affect promoter function, but deletions which in- catabolite repressor. cluded these sequences resulted in a loss of promoter activity (data not shown). The promoter sequence is associated with directly and DISCUSSION invertedly repeated sequences which contribute to the bind- The primary structure of the repressor gene for the S. ing site for the lacR protein (the operator site) and the aureus lac operon has been determined. The LacR protein binding site of the catabolite repressor. The binding sites for shares many similarities with the E. coli DeoR protein. The the two transcriptional regulators overlap. We postulate that two proteins are similar in size (251 versus 252 residues) and catabolite repression of the staphylococcal lac operon in- sequence (26% overall homology, 57% homology in the volves the action of a negative effector catabolite repressor predicted helix-turn-helix regions), and both are transcrip- that binds to the promoter of the operon at a site which may tional repressors which bind inducing species that are phos- include the -60 repeat. This conclusion is reinforced by a phorylated compounds (galactose-6-phosphate versus deox- diminution of catabolite repression in cells bearing a multi- yribose-5-phosphate). The TTG codon at positions 1057 to copy plasmid bearing the -60 repeat sequence. The pres- 1059 was assigned as the initiation codon. This assignment ence ofthis sequence is thought to titrate a limited amount of was based largely on relative position to the ribosome- the catabolite repressor and thus allow leaky expression of binding-site sequence. All genes thus far analyzed in gram- the lac operon in the presence of glucose in the growth positive bacteria are preceded by a strong ribosome-binding- medium. Because a relatively small number of plasmid site sequence. The lacR ORF could actually be extended an copies can exert this effect, it is expected that this catabolite additional 12 codons upstream of the TTG to an in-frame repressor is specific for the lac operon and is not likely to be ATG. However, this ATG is not associated with a ribosome- a global regulator, which would likely be present in greater binding-site sequence and is therefore unlikely to be the amounts in the cell. The catabolite repressor is proposed to translational start codon. bind to the promoter region when glucose is present and We have been unable to demonstrate repressor function in prevent transcription of the operon. The identity of this E. coli host cells. When the lacR gene was fused to the E. negative regulator, the catabolite repressor, is under inves- coli lacZ determinant such that ,-galactosidase expression tigation. The mechanism of catabolite repression in gram- was dependent upon the staphylococcal transcription and positive bacteria, which is unlikely to involve cyclic AMP, is VOL. 172, 1990 REGULATION OF S. AUREUS lac 3811 largely unknown. Interestingly, catabolite repression of 9. Dandanell, G., and K. Hammer. 1985. Two operator sites amylase production and aconitase synthesis in B. subtilis are separated by 599 base pairs are required for deoR repression of to negative regulatory as well the deo operon of Escherichia coli. EMBO J. 4:3333-3338. postulated involve protein(s) 10. Dodd, I. B., and J. B. Egan. 1987. Systematic method for the (11, 23, 36). However, in the amylase system, the binding detection of potential X cro-like DNA binding regions in pro- site for the catabolite repressor is downstream of the tran- teins. J. Mol. Biol. 194:557-564. scription start site, not within the promoter region. Se- 11. Fouet, A., and A. L. Sonenshein. 1990. A target for carbon quences between positions -84 and -67 compose at least source-dependent negative regulation of the citB promoter of part of the proposed target site for catabolite repression of Bacillus subtilis. J. Bacteriol. 172:835-844. citB (11). Whether there is a mechanism or mechanisms 12. Guyer, M. S. 1978. The -yb sequence of F is an insertion common to gram-positive bacteria for catabolite repression sequence. J. Mol. Biol. 126:347-365. or whether species-specific forms of regulation exist await 13. Guyer, M. S. 1983. Uses of the transposon y8 in the analysis of studies. cloned genes. Methods Enzymol. 101:362-369. further 14. Hengstenberg, W., W. K. Penberthy, and M. L. Morse. 1970. aureus operon was cloned by the creation of a The S. lac Purification of the staphylococcal 6-phospho-p-D-galactosidase. transposon Tn551 insertion-generated lactose constitutive Eur. J. Biochem. 14:27-32. mutant (6). The insertion site of TnSSl has been identified in 15. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for this report (Fig. 4). The insertion of the transposon was preparation of bacterial plasmids. Anal. Biochem. 114:193-197. between base positions 1963 and 1964 in the sequence. This 16. Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O'Reilly, P. M. site is downstream of lacR and upstream of the promoter- Schlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic operator sequences of the structural genes of the operon. shock syndrome exotoxin structural gene is not detectably The transposon-containing clones of this region compete as transmitted by a prophage. Nature (London) 305:709-712. repressor binding as do the same clones 17. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and effectively for efficient site-specific mutagenesis without phenotypic selection. lacking the TnSSJ insertion (data not shown). Therefore, the Methods Enzymol. 154:367-382. presence of the transposon does not interfere with repressor 18. Liu, L., W. Whalen, A. Das, and C. M. Berg. 1987. Rapid binding to the operator. The most likely explanation for the sequencing of cloned DNA using a transposon for bidirectional constitutive expression of the lac genes is that the transpo- priming. Nucleic Acids Res. 22:9461-9470. son interferes with expression of lacR. Promoter activity 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular originating within the transposon and extending outward cloning: a laboratory manual. Cold Spring Harbor Laboratory, from TnSSJ backward across lacR might negatively affect Cold Spring Harbor, N.Y. lacR transcription. The lacR gene, which is expressed at low 20. McClatchy, J. K., and E. D. Rosenblum. 1963. Induction of levels, may have a promoter which is a poor competitor lactose utilization in Staphylococcus aureus. J. Bacteriol. 86: 1211-1215. against a countertranscript from a stronger promoter. 21. Miller, J. H. 1972. Experiments in molecular genetics. Cold ACKNOWLEDGMENTS Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Morse, M. L., K. L. Hill, J. B. Egan, and W. Hengstenberg. We thank Everett Rosey for assistance with the computer pro- 1968. Metabolism of lactose by Staphylococcus aureus and its grams and for helpful discussions and Charles P. Moran, Jr., for genetic basis. J. Bacteriol. 95:2270-2274. pLC4. 23. Nicholson, W. L., Y.-K. Park, T. M. Henkin, M. Won, M. J. This work was supported by University of Kansas General Weickert, J. A. Gaskell, and G. H. Chambliss. 1987. Catabolite Research Fund Allocation 3190-XX-0038 and by a Biomedical repression-resistant mutations of the Bacillus subtilis alpha- Sciences Support Grant administered by the University of Kansas amylase promoter affect transcription levels and are in an (4831-0711-3). operator-like sequence. J. Mol. Biol. 198:609-618. LITERATURE CITED 24. Oskouian, B., and G. C. Stewart. 1987. Cloning and character- 1. Band, L., and D. J. Henner. 1984. Bacillus subtilis requires a ization of the repressor gene of the Staphylococcus aureus "stringent" Shine-Dalgarno region for gene expression. DNA lactose operon. J. Bacteriol. 169:5459-5465. 3:17-21. 25. Postma, P. W., and J. W. Lengeler. 1985. Phosphoenolpyruvate: 2. Bisset, D. L., and R. L. Anderson. 1973. Lactose and D- carbohydrate phosphotransferase system of bacteria. Micro- galactose metabolism in Staphylococcus aureus: pathway of biol. Rev. 49:232-269. D-galactose 6-phosphate degredation. Biochem. Biophys. Res. 26. Ray, C., R. E. Hay, H. L. Carter, and C. P. Moran, Jr. 1985. Commun. 52:641-647. Mutations that affect utilization of a promoter in stationary- 3. Blumenthal, H. J. 1972. Glucose catabolism in staphylococci, p. phase Bacillus subtilis. J. Bacteriol. 163:610-614. 111-135. In J. 0. Cohen (ed.), The staphylococci. John Wiley & 27. Reddy, P., A. Peterkofsky, and K. McKenney. 1985. Transla- Sons, Inc., New York. tional efficiency of the E. coli adenylate cyclase gene: mutating 4. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementa- the UUG initiation codon to GUG or AUG results in increased tion analysis of the restriction and modification of DNA in gene expression. Proc. Natl. Acad. Sci. USA 82:5656-5660. Escherichia coli. J. Mol. Biol. 41:459-472. 28. Reizer, J., J. Deutscher, F. Grenier, J. Thompson, W. Hengsten- 5. Breidt, F., Jr., W. Hengstenberg, U. Finkeldei, and G. C. berg, and M. H. Saier, Jr. 1988. The phosphoenolpyruvate: Stewart. 1987. 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McClure, and J. P. Houchins. 1985. A 31. Scheer-Abramowitz, J., T. J. Gryczan, and D. Dubnau. 1981. rapid single-stranded cloning strategy for producing a sequential Origin and mode of replication of plasmids pE194 and pUB110. series of overlapping clones for use in DNA sequencing: appli- Plasmid 6:67-77. cation to sequencing the corn mitochondrial 18S rDNA. Plasmid 32. Shapira, S. K., J. Chou, F. V. Richaud, and M. J. Casadaban. 13:31-40. 1983. New versatile plasmid vectors for expression of hybrid 3812 OSKOUIAN AND STEWART J. BACTERIOL.

proteins coded by a cloned gene fused to lacZ gene sequence 35. Valentin-Hansen, P., P. Hojrup, and S. Short. 1985. The primary encoding an enzymatically active carboxy-terminal portion of structure of the DeoR repressor and Escherichia coli K12. 3-galactosidase. Gene 25:71-82. Nucleic Acids Res. 13:5927-5936. 33. Sullivan, M. A., R. E. Yasbin, and F. E. Young. 1984. New 36. Weickert, M. J., and G. H. Chambliss. 1989. Genetic analysis of shuttle vectors for Bacillus subtilis and Escherichia coli which the promoter region of the Bacillus subtilis a-amylase gene. J. allow rapid detection of inserted fragments. Gene 29:21-26. Bacteriol. 171:3656-3666. 34. Valentin-Hansen, P., H. Aiba, and D. Schumperli. 1982. The 37. Yanisch-Perron, C., J. Viera, and J. Messing. 1985. Improved structure of tandem regulatory regions in the deo operon of M13 phage cloning vectors and host strains: nucleotide se- Escherichia coli K12. EMBO J. 1:317-322. quence of the M13mpl8 and pUC19 vectors. Gene 33:103-119.