JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4641-4651

Vol. 172, No. 8

0021-9193/90/084641-11$02.00/0 Copyright C 1990, American Society for Microbiology

Peptidase D Gene (pepD) of Escherichia coli K-12: Nucleotide Sequence, Transcript Mapping, and Comparison with Other Peptidase Genes BERNHARD HENRICH,l* URSULA MONNERJAHN,1'2 AND ROLAND PLAPP1 Fachbereich Biologie, Abteilung Mikrobiologie, Universitat Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern,' and Institut fur Genetik, Universitat KoIn, Weyertal 121, D-5000 Cologne 41,2 Federal Republic of Germany Received 18 January 1990/Accepted 30 May 1990

The nucleotide sequence of a 2.3-kilobase-pair DNA fragment of Escherichia coli that contains the transcription signals and the coding region of the pepD gene specifying aminopeptidase D was determined. The location and extent of the open reading frame were verified by partial amino acid sequencing of the purified pepD product. By use of a promoter-screening vector, initiation signals for pepD transcription were located in the 5'-flanking region of the open reading frame. Analysis of pepD transcripts by Si mapping, primer extension, and Northern (RNA) hybridization revealed two species of monocistronic mRNA with different 5' ends and a common 3' end. Calculation of the degree of codon usage bias in the coding region suggested that the efficiency of pepD translation is relatively low. As deduced from the predicted amino acid sequence, peptidase D is a slightly hydrophilic protein of 485 amino acid residues that contains no extended domains of marked hydrophobicity. Structural and functional features of the pepD gene are discussed and compared with other already sequenced peptidase genes of E. coli. The characterization of mutant strains has allowed the identification of at least six peptide-hydrolyzing enzymes in Escherichia coli (17, 22) that are similar to the peptidases of the closely related organism Salmonella typhimurium with respect to their substrate specificities. Peptidases play essential roles in the utilization of peptide substrates supplied in the medium (33) as well as in the breakdown of intracellular proteins, especially under conditions of nutritional starvation (39). The investigation of biochemical properties of these enzymes and of their regulation could be facilitated by the availability of the nucleotide sequences of the corresponding genes. However, structural and functional analysis of peptidase genes was only initiated during the last few years. Meanwhile, the sequences of the E. coli peptidase genes pepN, which seems to be regulated by phosphate and oxygen limitation (1, 5), pepA, which is identical to xerB (34), and pepP, which is a proline-specific aminopeptidase (40), have been published. Recently, we were able to clone another peptidase gene, pepD, taking advantage of the unique specificity of peptidase D for the unusual dipeptide 0-alanyl-L-histidine (14). The exact map position of the pepD gene on the E. coli chromosome was determined by sequence analysis of its upstream and amino-terminal regions, which revealed the contiguity of pepD and the adjacent gpt locus (10). In this report, we present the complete DNA nucleotide sequence of the pepD gene and of its 3'-flanking region and identify the probable initiation and termination sites of pepD-specific transcription. Properties that can be inferred from the sequence are discussed and related with features of the pepN, pepA, and pepP products.

eral Republic of Germany); strain NK5526 (hisG:: TnJO IN[rrnD-rrnE]J) was provided by N. Kleckner (Harvard University, Cambridge, Mass.). Plasmid pDS5 (35) was from H. Bujard (Zentrum fur Molekularbiologie, Heidelberg, Federal Republic of Germany). Constructions of the peptidase D-deficient strain UK61 and of plasmid pJK13 have been described elsewhere (14). Recombinant DNA techniques. Restriction enzymes and other nucleic acid-modifying enzymes were used as recommended by the manufacturers. Plasmids were isolated, and DNA fragments were isolated from gels by the methods of Maniatis et al. (19). Phage M13 DNA was isolated by the method of Messing et al. (20). Determination of amino acid sequences. Peptidase D, purified from strain UK61(pJK13) (10), was digested with trypsin and subsequently subjected to high-pressure liquid chromatography. The amino-terminal regions of selected fragments producing individual peaks in the elution profile were sequenced by automated Edman degradation on a gas-phase protein sequencer including on-line identification of the phenylthiohydantoin derivatives (6). Generation of deletions and DNA sequencing. As the 2.4kilobase insert of pJK13 that carries the pepD gene (10) was subject to spontaneous deletions after insertion into phage M13 vectors, two subfragments (a 1.0-kilobase EcoRI-SalI fragment and a 1.4-kilobase SalI-PstI fragment) were cloned into M13mpl8 and M13mpl9, respectively. Dideoxy sequencing of DNA was performed as described previously (10), with the use of a series of M13 subclones carrying overlapping deletions in the respective inserts of chromosomal DNA. Targeted deletions were generated by the exonuclease III method of Henikoff (9). Construction of plasmid pJK13t,. To prevent pepD transcription from the lac promoter that precedes the pepD gene in the pUC18 derivative pJK13 (14), we constructed plasmid pJK13t1. A 707-base-pair (bp) PvuII-XbaI fragment containing the terminator t, from the vector pDS5d (see Fig. 2A) was isolated and, after filling in the XbaI ends with the

MATERIALS AND METHODS Bacterial strains, bacteriophages, and plasmids. E. coli K-12 JM109 (38) and phage M13mpl8 and M13mpl9 (24) were obtained from Pharmacia LKB GmbH (Freiburg, Fed*

Corresponding author. 4641

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HENRICH ET AL.

Klenow fragment of DNA polymerase I, was inserted into the likewise filled-in EcoRI site of pJK13. As verified by nuclease S1 mtapping, the resulting plasmid, pJK13tj, directed only the synthesis of pepD transcripts that were initiated at the authentic pepD promoters. Isolation of RNA. Total RNA was prepared from E. coli by the cold phenol extraction method (12) as modified by Sawers and Bock (28). After the final precipitation step, RNA was not centrifuged and dried but was stored in ethanol at -70°C. The RNA concentration was determined by measuring the A260 after centrifugation of a portion of this suspension and dissolution of the sediment in distilled water (25 A260 units 1 mg of RNA per ml). Depending on the strain, a 100-mi culture of bacteria yielded 0.5 to 1.0 mg of RNA. RNA preparations were analyzed on 1% agarose gels before use. Preparation of radioactive probes. To map the initiation sites of pepD transcripts, we used the 321-bp Hinfl fragment derived from the recombinant plasmid pJK13 (see Fig. 2B) as a probe. This fragment, comprising 168 amino-terminal nucleotides of the pepD coding region, was dephosphorylated and labeled at its 5' ends by using T4 polynucleotide kinase (19). For the analysis of the 3'-flanking region of pepD, a 505-bp RsaI fragment, including 226 nucleotides of the pepD coding sequence (see Fig. 2B), was labeled at its 3' ends with [a-32P]dTTP by using T4 DNA polymerase as described by Hartmann and Erdmnann (7), with the exception that 360 ng of the DNA fragment was treated with 2.5 U of T4 DNA polymerase. Labeled fragments were separated from free nucleotides by passage over 2-ml Sephadex G-50 columns equilibrated with 10 mM Tris hydrochloride (pH 7.6)-i mM EDTA. Peak fractions were pooled, precipitated with 3 volumes of ethanol, and stored in ethanol at -70°C. Alternatively, uniformly labeled single-stranded probes complementary to the proposed 5' end (nucleotides -153 to +168) and to an internal part (nucleotides +169 to +505) of pepD mRNA (see Fig. 2B) were synthesized by a modification of the strategy described by Burke (3). The corresponding tenmplates, M13Hinf321 and M13Hinf337, had been constructed by insertion of the 321- and 337-bp Hinfl fraginents of plasmid pJK13 into the HincIl site of M13mpl8 replicative-form DNA. Single-stranded M13Hinf321 DNA was annealed with a 25-mer synthetic oligonucleotide, 5'-CCC ACAGCGGCTGTGGAGATAATTG-3', that had been synthesized on a Pharmacia Oene Assembler Plus and purified by high-pressure liquid chromatography. Single-stranded M13Hinf337 DNA was annealed with the 17-mer lac universal primer. Annealing was done in volumes of 20 pI containing 1 ,g of template DNA, 10 ng of primer, 10 mM Tris =

hydrochloride (pH 7.6),

10 mM

MgCl2,

and 2 mM dithiothre-

itol at 650C for 10 mim with slow cooling to room temperature. The primers were extended in the same buffer supplemented with unlabeled dCTP, dGTP, dTTP (each at 40 ,uM), 25 pmol (25 pCi) of [a-35S]dATP, and 3 U of DNA polymerase (Klenow fragment). The mixtures (30 p1) were incubated at 370C for 15 min and chased for a further 15 min after addition of unlabeled dATP to a final concentration of 40 ,uM. Probes were generated by subsequent digestion of double-stranded DNA regions with Ba#tHI followed by purification of the appropriate single-stranded fragments on sequencing gels containing 6%o polyacrylamide and 7% urea. Nucease Si protection analysis. To identify the ends of pepD mRNAs by nuclease S1 mapping, we modified the method of Berk and Sharp (2) as follows. RNA samples (100

to 120 pg) and about 20 fmol of the respective end-labeled probe fragments were coprecipitated, dried for 17 min at room temperature, redissolved in 30 pl of hybridization buffer containing 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 6.4), 1 mM EDTA, 0.4 M NaCI, and 80% formamide, and overlaid with a small drop of paraffin. The samples were denatured at 85°C for 15 min, rapidly transferred to the appropriate hybridization temperatures, and incubated for 15 h. To digest the remnaining singlestranded nucleic acids, the mixtures were diluted with 270 ,ul of ice-cold S1 buffer containing 55.6 mM sodium acetate (pH 4.6), 311 mM NaCl, 5 mM ZnSO4, single-stranded salmon testes DNA (20 p,g/ml), and nuclease S1 (480 U/mi) and incubated at 37°C for 45 mn. After the addition of 5 p,g of carrier yeast tRNA, the nucleic acids were extracted with phenol-CHCl3-isoamyl alcohol (25:24:1), precipitated with ethanol, and dissolved in 8 p1 of formamide dye (19). Portions (2 pa) of these samples were heated at 95°C for 3 min and subjected to electrophoresis in polyacrylamide sequencing gels. For better resolution of the 5' ends of pepD transcripts, alternatively, a single-stranded probe (5 x 10' cpm) synthesized by extension of a synthetic primer was used in S1 protection experiments as described above. Primer extenson nalysis. To determine the 5' end of pepD mRNA by primer extension, we used the same synthetic oligonucleotide (25-mer) as in the synthesis of a singlestranded DNA probe (see above). The oligonucleotide (3 pmol) was labeled at its 5' end with 9 pmol (45 ,uCi) of (-Y32p] ATP by T4 polynucleotide kinase (36) followed by passage over a 2-ml Sephadex G-25 column equilibrated in water. The primer extension experiment followed a previously described protocol (13) with some modifications. Portions (0.7 pmol) of labeled oligonucleotide were frozen in liquid nitrogen and lyophilized. Samples of RNA (10 pg) were precipitated with ethanol, dried for 15 min at room temperature, and redissolved in 16.5 pl of hybridization buffer (see above). These RNA solutions were used to suspend the dry pellets of labeled ofigonucleotide. Annealing was allowed to proceed for 8 h at 370C followed by the addition of 93.5 pl of 0.3 M sodium acetate and ethanol precipitation. The washed and dried pellets were dissolved in 10 IlI of water and subsequently combined with 300 units of Moloney murine leukemia virus reverse transcriptase in a 20.jd reaction volume containing 50 mM Tris hydrochloride (pH 8.3), 8 mM MgC12, 30 mM KCl, 10 mM dithiothreitol, and 0.5 mM each of all four deoxynucleoside triphosphates. The reaction was performed for 40 min at 43°C and was followed by extraction of the nucleic acids with phenol-CHCl3-isoamyl alcohol (25:24:1) and ethanol precipitation. The sediments were dissolved in 4 plA of water with 8 pl of formamide dye (19) and heated at 95°C for 3 min. Portions (3 pi) of these samples were analyzed on an 8% sequencing gel. Norther (RNA) blot analysis. Total RNA (5 pg) was separated on a 1.2% agarose-1.1%o formaldehyde gel and transferred to a nitrocellulose membrane by the method of Selden (29). rRNA bands and molecular weight markers were visualized by ethidium bromide staining in a piece of the gel that had been cut off before the transfer. Prehybridization, hybridization, and washings of the membrane filter were done by the methods of Maniatis et al. (19). To detect pepD-specific transcripts, we used a 35S-labeled probe (107 cpm) synthesized by primer extension on an M13 template. Compter analyses. The programs of the Microgenie menu (Beckman Instruments, Inc., Palo Alto, Calif.) were used to analyze nucleotide and amino acid sequences.

VOL. 172, 1990

SEQUENCE AND TRANSCRIPTION OF E. COLI pepD GENE

Materials. Restriction endonucleases and nucleic acidmodifying enzymes were purchased from Boehringer GmbH (Mannheim, Federal Republic of Germany) or Pharmacia LKB GmbH. [a-35S]dATP (600 or 1,000 Ci/mmol), [-y-32p] ATP (3,000 or 5,000 Ci/mmol), and [a-32P]dTTP (3,000 Ci/ mmol) were obtained from Amersham GmbH (Braunschweig, Federal Republic of Germany). Ultrapure deoxynucleoside triphosphates and the 17-mer lac universal primer were from Pharmacia LKB GmbH. Nitrocellulose membranes were purchased from Schleicher & Schuell GmbH (Dassel, Federal Republic of Germany). RESULTS Nucleotide sequence analysis of pepD gene. A partial restriction map of the recombinant plasmid pJK13 that carries a functional pepD gene has been established (10). The nucleotide sequence of both strands of a 2,333-bp segment of this plasmid was determined mainly by using exonuclease IIInuclease Si-generated deletion subclones. Some areas for which the appropriate deletions were not obtained were sequenced after subcloning of individually isolated small restriction fragments. The nucleotide sequence of the pepD gene and surrounding regions is presented in Fig. 1 together with the amino acid sequence predicted for the coding region. The DNA sequence from nucleotide -186 to nucleotide 168 is identical to the previously established sequence of the 5'-flanking and amino-terminal regions of the pepD gene (10). The size of the open reading frame beginning at a GTG initiation codon at nucleotide 1 and extending to the TAA stop codon at nucleotide 1458 is sufficient to encode a protein of 485 amino acids with a deduced molecular mass of 52.89 kilodaltons. This value agrees closely with the 52-kilodalton molecular mass of peptidase D as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14). The start codon of the pepD gene has been unambiguously identified by amino acid sequence determination of the first 12 residues of purified peptidase D (10). To verify the extension and the proper reading frame of pepD, we isolated and partially sequenced three tryptic fragments of peptidase D. The amino acid sequences of these peptides correspond to parts of the deduced polypeptide sequence and are indicated in Fig. 1. Screening for pepD promoter. In a recent work, we demonstrated that the 5' ends of the pepD coding region and the adjacent gpt locus are spaced by only 260 bp (10). As pepD and gpt are oriented in opposite directions, and the existence of an intermediate gene has been excluded, it is obvious that pepD-specific transcription must be initiated in the inter-

genic region. To achieve a rough localization of the pepD promoter, we examined appropriate restriction fragments of the recombinant plasmid pJK13 containing different portions of the gpt-pepD intergenic sequence (Fig. 2B) for promoter activity by use of the promoter-screening vector pDS5d (Fig. 2A). After insertion in the HinclI site of pDS5d, the 321-bp Hinfl fragment allowed the growth of nearly the same number of chloramphenicol-resistant transformants as the 794-bp AluI fragment. Thus, the DNA region upstream of the left end of the 321-bp Hinfl fragment in Fig. 2B, corresponding to nucleotide -153 (Fig. 1), does not significantly contribute to the activity of the pepD promoter. As this DNA region contains the Pribnow box of the gpt gene (26), it appears that the functional elements of the gpt and pepD promoters do not overlap. On the contrary, the 685-bp HincII fragment,

4643

starting at position -68, after cloning into pDS5d, yielded only 2.5% of the chloramphenicol-resistant transformants that had been obtained with the 794-bp AluI fragment. The DNA segment between positions -68 and -153 consequently must contain signals that are indispensable to efficient initiation of pepD transcription. Mapping of pepD transcripts. To define the limits of pepD transcription and to identify the corresponding promoter and terminator signals, we mapped the endpoints of the major pepD-specific transcripts by nuclease Si protection experiments, by primer extension analysis, and by Northern blot hybridization. (i) 5'-endpoint determination. High-resolution S1 protection experiments were performed with the 321-bp Hinfl

fragment of pJK13, containing the functional pepD promoter region (Fig. 2B). To avoid S1 artifacts which may result from hyperexpression of high-copy-number genes, we obtained the mRNA from strain NK5526, which contains a single chromosomal copy of the pepD gene. This RNA, after hybridization with the probe, protected two DNA fragments of 262 + 2 nucleotides and 230 ± 2 nucleotides from nuclease S1 digestion (Fig. 3A). This indicates the existence of two species of pepD-specific mRNA which differ in length by about 32 nucleotides at their 5' ends. As these 5' endpoints were evident in eight different experiments with different RNA preparations and different probes (Fig. 3B), it is unlikely that their detection is due to artifacts associated with S1 digestion or RNA isolation. The accuracy of Si mapping was also confirmed by primer extension analysis (Fig. 3C). In this case, mRNA from strain NK5526 was hybridized to an oligonucleotide primer complementary to nucleotides 16 to 40 (Fig. 1). We detected two major cDNAs with apparent endpoints at positions -91 or -93 and -60, respectively, which are in excellent agreement with the approximate 5' ends of pepD mRNA resolved by Si mapping. Thus, the results of the two techniques are consistent with two transcriptional start sites, as indicated in Fig. 1. However, the possibility that one or perhaps both of the 5' endpoints arose by processing of a longer transcript cannot be denied. Analysis of the nucleotide sequence upstream of the two potential initiation sites for pepD transcription revealed several elements that may serve as possible recognition and binding signals for RNA polymerase (27). For transcriptional start site 1 (Fig. 1), this confirms our previous suggestion that the sequence about 100 nucleotides upstream from the pepD start codon is involved in promotion of pepD expression (10). (ii) 3'-endpoint determination. A sequence displaying typical features of a p-independent transcriptional terminator (27) is centered around nucleotide 1486 in Fig. 1. It has the potential to form a G+C-rich stem-loop structure with a free energy of -32 kcal/mol and is followed by a run of three Us (Fig. 4B). The physiological role of this structure was verified by high-resolution nuclease Si protection analysis of RNA prepared from strain NK5526, using the 505-bp RsaI fragment of plasmid pJK13 (Fig. 2B) as a probe. The upper, more intense signals in Fig. 4A most probably resulted from transcription termination 20 + 3 nucleotides beyond the center of the dyad symmetry of the predicted stem-loop structure (Fig. 4B). The lower bands originate from probe fragments that end at different positions within the loop of the hairpin. They are suspected to be artifacts generated by nuclease Si cleavage within the unpaired region of an analogous hairpin structure which, due to its high AG value, may also form during RNA-DNA hybridization.

SPz. sv

-180

-170

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-200 5'...TGCGCGAGATT7ATAGAGATCTGGCGCACTAAAAACCAGTATTTCACATGAGTCCG

3'...ACGCGCTCTAATATCTCTAGACCGCGTGA... 5' 21-35a CGT

1/-35-

-140

1/-35b

*RNAl

J2 Il-l1a

AM72 ssuA XTTACGCACSCCCCGCCGG

2/-lOb RA2 | -30 _40 pho-box7 CGCAAGC T,oTCTATATGTAAACA MnTGCTAACCCTGTGACCTGCAATACTGTTT

21- 10a

-20

_~~~50

pp

SD D

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T(;CGGGTGATCGACRAAGAGACTTAACGTGTCTGAA,CTGTCTCAATTATCTCCACAGCCG

Met*rG1luL.uS@rGlnZ4uS-rProG1nPro

50

60

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40 CTGTGGGATATTTTTGCCAAAATCTGTTCTATTCCTCACCCGTCCTATCATGAAGAGCAA

T,.uTr

spI1ePheAlaLysI1eCyBSerI1eProHisProSerTyrHisGluGluGln 110

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100 CTCGCTGAATACATTGTTGGTTGGGCAAAAGAGAAAGGTTTCCATGTCGAACGCGATCAG LeuAlaGluT2yrIleVa.lGlyTrpAlaLysGluLyaG.lyPheH.isValGluArgAspGln 210 200 190 180 170 160 GTAGGTAATATCCTGATTCGTAAACCTGCTACCGCAGGTATGGAAAATCGTAAACCGGTC

ValGlyAsnIleLeuI.leArgLysProA.laThrAlaGlyMetGluAsnArgLysProVa1 230

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220 GTCTTACAGGCCCACCTCGATATGGTGCCGCAGAAAAATAACG,ACACCGTGCATGACTTC ValLeuGInAlaHisLeuAspMetValProGlnLy-2 __AphV1LA

280 ACGAAAGATCCTATCCAGCCTTATATTGATGGCGAATGGGTTAAAGCGCGCGGCACCACG

ThrLysAspProIlleGl nProTyrI leAspGl yGl uTrpVal LysAl aArgGl yThrThr 350

360

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39,0

340 CTGGGTGCGGATAACGGCATTGGTATGGCCTCTGCGCTGGCGGTTCTGGCTGACGAAAAC

LeuGlyAlaAspAsnGlyIleGlyMetAlaSerAlaLeuAlaValLeuAlaAspGluAsn 420

430

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450

410 400 GTGGTTCACGGCCCGCTGGAAGTGCTGCTG,ACCATGACCGAAGAAGCCGGTATGGACGT ValzValHisGlyProLeuGluValLeuLeuThrMetThrGluGluAlaGIy etAspGly 510 500 490 480 470 460 GCGTTCGGCTTACAGGGCAACTGGTTGCA,GGCTGATATTCTGATTAACACCGACTCCGAA

AlaPheGlyLeuGlnGlyAsnTrpLeuGlnAlaAspIeLeuIleAsnThrAspSerGlu 570 560 550 540 530 520 GAA,GAAGGTGAAATCTACATGGGTTGTGCGGGGGGTATCGACTTCACCTCCAACCTGCAT GluGluGlyGlPulleTyrMetGlyCysAlaGlyGlyIleAspPheThrSerAsnLeuH.is 630 620 610 600 590 580 TTAGATCGTGAAGCGGTTCCAGCTGGTTTTGAAACCTTCAAGTTAACCTTAAAAGGTCTG

_.

.ZeuAspArgGluAlaValProAlaGlyPheGluThrPheL sLeuThrLeuLysGlyLeu 690 680 670 660 650 640 AAAGGCGGTCACTCCGGCGGGGAAATCCACGTTGGGCTGGGTAATGCCAACAAACTGCTG

LysGlyGlyHisSerGlyGlyGluIleHiLsValGlyLeuGlyAsnAlaAsnLysLeuLeu 720

730

740

750

710 700 GTGCGCTTCCTGGCGGGTCATGCOGAAGAACTGGATCTGCGCCTTATCGATTTCAACGGC

y ValArcjEh-LuI-aGlyRinAloGluGluLou bspLeuArgLeuIlleAspPheAsnGl residue of the GTG first the to relative is Numbering

FIG. 1. Nucleotide sequence encompassing the pepD gene and its flanking regions. the divergently start codon of pepD, which is taken as +1. The segment up to position -187, containing the Pribnow box (gpt-10) of of chro.mosomal insert the to shown was overlap which (25) and Niuesch Schuimperli by the from is published sequence transcribed gpt gene DNA in (10). Boxed bases with angled arrows indicate the 5' endpo'ints (mRNA1, mRNA2) and 3' endpoint (stop) of the two most

pJK13 and -10 regions of the putative abundant mRNA species detected by S1 mapping and primer extension (see Fig. 3 and 4). Potential -35 2/-10a and 2/-10b for promoter 2/-35a transcniption initiation sites are denoted 1/-35a 1/-10a .and 1/-35b 1/-10b for promoter site 1- and inverted repeat site 2. A potential Shine,Dalgarno (32) sequence (SD) and a pho box-like sequence, (positions 91 to - 74) are indicated. Antne ot pepv coding the beginning marks arrow open The underlined. is 1502) to 1471 (positions structure capable of forming a stem-loop sequences region. The deduced amino acid sequence of peptidase D is given below the nonsense strand of the pepD gene. Boxed aminotoacid were corroborated by sequencing the amino termini of purified fragments of peptidase D. The sequence has been submitted the GenBank database under accession number M34034. 4644

VOL. 172, 1990

SEQUENCE AND TRANSCRIPTION OF E. COLI pepD GENE 760

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GGCACACTGCGTAACGCCATCCCGCGTGAAGCCTTTGCGACCATTGCTGTCGCAGCTGAT GlyThrLeuArgAsnAlaIleProAr JPluAlaPheAlaThrIleAlaValAl 9laAsp

AAAGTCGACGTCCTGAAATCTCTGGTGAATACCTATCAGGAGATCCTGAAAAACGAGCTG LysValAspValLeuLysSerLeuValAsnThrTyrGlnGluIleLeuLysAsnGluLeu GCAGAAAAAGAGAAAAATCTGGCCTTGTTGCTGGACTCTGTAGCGAACGATAAAGCTGCC AlaGluLysGluLysAsnLeuAlaLeuLeuLeuAspSerValAlaAsnAspLysAlaAla CTGATTGCGAAATCTCGCGATACCTTTATTCGTCTGCTGAACGCCACCCCGAACGGTGTG

LeuXleAlaLysSerArgAspThrPhelleArgLeuLeuAsnAlaThrProAsnGlyVal 1000

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ATTCGTAACTCCGATGTAGCCAAAGGTGTGGTTGAAACCTCCCTGAACGTCGGTGTGGTG IleArgAsnSerAspValAlaLysGlyValValGluThrSerLeuAsnValGlyValVal ACCATGACTGACAATAACGTAGAAATTCACTGCCTGATCCGTTCACTGATCGACAGCGGT

TyrMetThrAspAsnAsnValGlulleHisCysLeulleArgSerLeuIleAspSerGly 1120

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AAAGACTACGTGGTGAGCATGCTGGATTCGCTGGGTAAACTGGCTGGCGCGAAAACCGAA LysAspTyrValValSerMetLeuAspSerLeuGlyLysLeuAlaGlyAlaLysThrGlu GCGAAAGGCGCATATCCTGGCTGGCAGCCGGACGCTAATTCTCCGGTGATGCATCTGGTA

AlaLysGlyAlaTyrProGlyTrpGlnProAspAlaAsnSerProValMetHisLeuVal 1240

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CGTGAAACCTATCAGCGCCTGTTCAACAAGACGCCGAACATCCAGATTATCCACGCGGGC

ArgGluThrTyrGlnArgLeuPheAsnLysThrProAsnIleGlnlleIleHisAlaGly 1300

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CTGGAATGTGGTCTGTTCAAAAAACCGTATCCGGAAATGGACATGGTTTCTATCGGGCCA

LeuGluCysGlyLeuPheLysLysProTyrProGluMetAspMetValSerIleGlyPro 1360

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ACTATCACCGGTCCACACTCTCCGGATGAGCAAGTTCACATCGAAAGCGTAGGTCATTAC

ThrIleThrGlyProHisSerProAspGluGlnValHisIleGluSerValGlyHisTyr 1420

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TGGACACTGCTGACTGAACTGCTGAAAGAAATTCCGGCGAAGTAATTATTTGATTTGCTG TrpThrLeuLeuThrGluLeuLeuLysGlulleProAlaLysEnd stem-loop

CCGGATGGCGTTTAATCGCCTTCCGGCAG

1520

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~~CTTCATTATCCTTCGATAAAAGCC

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ATCCCTGTAAATGTCCGTCGCGGGTTGCCACGTTCAATCTGGTGATGGAACATTCGCCGC

TGCGATTTCAGCGCCGCGCTATTTTCCTGTTGCTGTTGCTCCAGCTTCCAGGCAATCAGC AATCGTGCCAGCCGCTTGTTAGCATGCTGACTACGCTCTGACTGAACCTTCACGCTAATA CCGGATGCCAAATGCGTGGCGCGTACCGCCGAGTCGGTTTTATTGACATGTTGACCGCCC GGCCCCGACGAACGCAGCGTCTCATAACGGATTGCATCCGATTGTTCCTGCTCATCAGCG

GTAAAACGCCCAATGCCCAGAAACCAGTTTTTGCGCCCATGATGAGGCCGATACGGACTC GGACAAATCCACTGAATAGTGCCACACCACTTTCGCTTAATGCCCATGCGTTATCGCCAT CGAGAGAAACCAGCGCCGAACGCAGTGTGTCAGAGTAGCGGCCCGTTTCTGTTTCCAGCA

CCGTTACCGCGACGTCTTGCCGGGTAAAAACGACCAACACAGAAGAAGCTGCGCCAATCG GGTAGCCGCGTCCGGGGTGTAAATCTGGCATCCCCACTACGCGCTGCATGTT... 3' FIG. 1-Continued.

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4646

B

a

HincII(685)

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Hinf 1(321) AluI (794)

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]PR

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FIG. 2. (A) Organization of the promoter-screening vector pDS5d. To construct pDS5d, the PN2S5IaC promoter was eliminated from plasmid pDS5 by XhoI-EcoRI digestion, filling in the ends with Klenow fragment, and religation. In pDS5d, the promoter-free cat gene still contains its original ribosome-binding site and is preceded by a multiple cloning site (mcs) with unique recognition sequences for SmaI, BamHI, Sall, HincIl, PstI, and HindIII. Eventual transcription of the cat gene is terminated at terminator t, of the rrnB operon of E. coli. To select recombinant plasmids containing functional promoters, we used chloramphenicol at a concentration of 10 g/ml. (B) Physical map of the pepD region of plasmid pJK13. The open reading frame of the pepD gene is indicated by the solid bar. The arrow shows the direction and extent of the two putative pepD transcripts (Fig. 1). FR, Fragments of plasmid pJK13 that were cloned into the promoter-screening vector pDS5d. PR, Fragments used as probes in nuclease Si mapping and Northern blot experiments. The lengths of the fragments (in base pairs) are given in parentheses. The scale of the map is 0.1 kilobase. Recognition sites for restriction enzymes are abbreviated as follows: E, EcoRI; A, AluI; R, RsaI; H, HincII; F, Hinfl.

(ui) Northern blot analysis. In an attempt to determine the lengths of pepD transcripts more directly, we performed Northern blots of total RNA which had been prepared from strain UK61 carrying plasmid pJK13t1. Owing to the presence of the plasmid, this RNA contains an increased fraction of pepD-specific mRNA (10). Blots of this RNA were hybridized to the uniformly labeled coding strand of the 337-bp Hinfl fragment of pJK13 which covers an internal part of the pepD structural region (Fig. 2B). This probe, within the resolution of the Northern blot experiments, detected one major class of pepD-specific transcripts in the range of 1,600 nucleotides (Fig. 5). This size agrees well with the calculated lengths of about 1,565 and 1,597 nucleotides of transcripts originating at the two putative pepD promoter sites and terminating at the predicted hairpin structure that follows the pepD coding region (Fig. 1).

Codon usage. Usually, the frequency of rare codons as defined by Konigsberg and Godson (15) in E. coli genes is about 4%. In the pepD coding region, in accordance with this compilation, rare codons occur as 4. 1% of the total codons in the reading frame and as 14.8 and 11%, respectively, in the nonreading frames. On the basis of the relative quantities of tRNAs in E. coli, Ikemura (11) established a list of codon preferences. We used this list to calculate the frequencies of optimal codon usage for the known sequences of peptidase genes. The pepD, -N, -A, and -P genes yielded values of 0.77, 0.72, 0.70, and 0.72, respectively. These values are indicative of middle-level-quantity proteins, present in less than 1,000 copies per cell (11). The most recent and most comprehensive analysis of available sequencing data (30) revealed a clear correlation between the degree of codon usage bias and the level of gene

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FIG. 3. Determination of the 5' ends of pepD transcripts. (A) Nuclease S1 mapping with 5'-end-labeled double-stranded 321-bp Hinfl fragment (Fig. 2B) as a probe. The probe was incubated with NK5526 RNA (lane 2), UK61 (ApepD) RNA (lane 3), and yeast tRNA (60 ,g) (lane 4) at 44.5°C, digested with nuclease S1, and electrophoresed. Lane 1, Undigested probe; lanes A to T, reference DNA sequencing ladder of M13mp18 DNA. The sizes of the reference bands (base pairs) corresponding to the protected probe fragments are indicated. They were calculated from the hybridization site of the lac primer. The start sites of pepD transcription were mapped by calculating the same number of bases from the 5' end of the probe. (B) Nuclease S1 mapping with uniformly labeled, single-stranded, 321-nucleotide Hinfl fragment as a probe. The probe was hybridized to NK5526 RNA (lane 1), digested with nuclease Si, and electrophoresed. Lanes A to T, Reference sequencing reactions on the M13Hinf321 template initiated by the same 25-nucleotide primer which was used to synthesize the probe (see Materials and Methods). Accordingly, the sequence is the complement of the one shown in Fig. 1. Sequences encompassing the 3' ends of the protected probe fragments (indicated by asterisks) are shown at the right of the panel. (C) Primer extension. The 25-mer oligonucleotide complementary to a sequence near the 5' end of the pepD gene was used to prime cDNA synthesis on RNA from strain NK5526 (lane 1). Reference sequencing reactions (lanes A to T) were initiated by the same primer. Sequences of the coding strand encompassing the 3' ends of the major extension products (indicated by asterisks) are shown at the left of the panel.

expression in E. coli. Calculation of the relative synonymous codon usage values (30) of pepD, -N, -A, and -P again suggested that the efficiency of peptidase synthesis in E. coli is relatively low. Hydrophathic character of peptidases. The predicted amino acid sequences of peptidases D, N, A, and P imply an overall hydrophilic character of these proteins. This supports the generally accepted assumption that most of the peptidases of E. coli are soluble proteins, which are exclusively located in the cytoplasm of the cells (17, 21). Table 1 shows that the overall hydrophobicities decrease in the order PepD-PepAPepP-PepN. Plotting of the average residue hydrophobicity as a function of residue number (16) showed considerable unsteadiness in the profiles of the four peptidases (Fig. 6), which is indicative of most soluble proteins. However, no obvious similarities between these profiles could be detected. Sequence similarities of peptidases. To compare the protein sequences of peptidases D, N, A, and P, we applied the dot-matrix method using a window of 10 amino acids and a stringency of 30%, but no identity of significant value was found. Computer-aided searches of protein sequence data banks also showed no striking similarity of peptidase D to

any sequence of other bacterial and eukaryotic peptidases and proteases contained in these banks. Only two proteins were detected that resemble peptidase D to a degree which, by calculation of the normalized alignment score (4), is at least of marginal significance. These proteins are the Sendai virus matrix protein (33.3% identity within a segment of 66 amino acids) and the human cytoskeletal keratin (31.5% identity within a segment of 73 amino acids).

DISCUSSION The coding region of the E. coli gene pepD was unequivocally assigned to a part of the established nucleotide sequence by oligopeptide sequencing of internal fragments of purified peptidase D. This enzyme, a broad-specificity peptidase, is a slightly acidic protein (59.6% of the amino acids are negatively charged) of Mr 52,890 (485 residues). Expression of pepD. Viewing the efficiency of pepD transcription initiation, nucleotides upstream of the 5' endpoints of the two putative mRNA species can be identified that display similarity to the -10 and -35 regions of the E. coli au70 consensus promoter (8). For pepD mRNA 1, two dif-

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ferent pairs of possible -10 and - 35 elements are denoted in Fig. 1. Although each of them is equally matched to the consensus, the -10 and -35 elements of the pair proximal to the initiation site of mRNA 1 are not appropriately spaced and thus may not function as an RNA polymerase-binding site. For pepD mRNA 2, two possible -10 regions with equal homology to the consensus Pribnow box were detected. The region proximal to mRNA start site 2, however, is not preceded by an obvious -35 sequence. All these elements are located on a 85-bp Hinfl-HincII fragment (Fig. 2B) that, by use of a promoter-screening vector, was found to contain essential promoter elements. As verified by nuclease Si and primer extension studies, this fragment covers promoter site 1 completely, whereas it lacks the mRNA initiation point of site 2 which is disconnected from the respective -35 and -10 regions by Hincll restriction at position -68. The effective role of promoter site 2 thus remains to be ascertained. Murgier and Gharbi (23) demonstrated that in E. coli, transcription of the pepN gene is stimulated by phosphate limitation. Accordingly, we found a threefold increase of

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FIG. 4. Termination of pepD transcripts. (A) Nuclease Si mapping with 3'-end-labeled 505-bp RsaI fragment (Fig. 2B) as a probe. The probe was incubated with NK5526 RNA (lane 2) and UK61 (ApepD) RNA (lane 3) at 46.5°C, digested with nuclease Si, and electrophoresed. Lane 1, Undigested probe; lanes A to T, reference sequencing ladder of M13mpl8 DNA. The sizes of the protected probe fragments (base pairs) were calculated as described in the legend to Fig. 3A. (B) Potential secondary structure of the RNA transcript in the 3'-flanking region of the pepD gene. Numbering of the nucleotides is as in Fig. 1. The stop codon of the pepD coding region is boxed. The 3' ends of transcripts, detected by nuclease S1 mapping, lie within the region indicated by the solid bar. The termination frequencies at the individual positions within this region, as estimated from Fig. 4A, are reflected by the thickness of the bar.

peptidase D activity, measured with P-alanyl-L-alanine as a specific substrate (37), under conditions of phosphate starvation. We therefore inspected the intergenic region between the genes gpt and pepD for signs of transcriptional regulation. The only particular feature detected was a sequence (nucleotides -91 to -74, Fig. 1) showing 61% identity to the consensus pho box (18) that is suspected to interact with the phoB product in the promoters of genes belonging to the pho regulon (18, 31). Thus, the PhoB protein may also be involved in a possible modulation of pepD transcription. Efficient termination of pepD transcription most probably occurs at a very stable stem-loop structure about 48 nucleotides beyond the stop codon. Therefore, pepD mRNA appears to be monocistronic. Cellular location of peptidases. As the amino acid sequence at the N terminus of purified peptidase D corresponds to the 5' end of the pepD coding region (10), amino-terminal processing of a precursor form of the enzyme must be excluded. This is consistent with the mainly cytoplasmic localization of E. coli peptidases (17, 21). However, we noticed that, by heavy overexpression of the pepD gene from plasmid pJK13, E. coli acquires the ability to utilize the substrate D-alanyl-L-leucine, which cannot be transported across the cytoplasmic membrane. Therefore, at least in this case, a significant portion of peptidase D activity appears to be located at the outside of the cytoplasmic membrane. We also found about 8% of peptidase A activity in the osmotic shock fluid of E. coli, whereas cytoplasmic marker enzymes could not be detected in this fraction. Thus,

SEQUENCE AND TRANSCRIPTION OF E. COLI pepD GENE

VOL. 172, 1990

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some of the hydrophobic regions indicated in Fig. 6, although not optimally suited to promote export or membrane localization of the respective proteins, may be involved in the translocation of a small fraction of some peptidases into or through the cytoplasmic membrane. A variety of useful functions of extracytoplasmically located peptidases can be imagined. These include the degradation of peptides which, due to their length or the presence of unusual amino acids, cannot be transported into the cells and the eventual reutilization of the signal peptides of secretory proteins. Common features of E. coli peptidases. Physiological and structural characteristics deduced from the available sequences of four E. coli peptidases (Table 1) include similar frequencies of optimal codon usage and nearly identical isoelectric points of about 6.0. The monomer molecular masses of peptidases D, A, and P are all about 50 kDa, whereas peptidase N is approximately twice this size. This led us to examine whether the pepN gene might originate from the duplication of a hypothetical precursor gene, but neither on the level of DNA sequence nor on the level of protein sequence could any significant homology between the amino- and carboxy-terminal moieties of the pepN gene or its product be detected. FIG. 6. Hydropathy profiles of peptidases D, N, A, and P. The primary structures of the peptidases were deduced from the respective nucleotide sequences. The hydropathy values, as calculated by the algorithm of Kyte and Doolittle (16), were multiplied by 10 and averaged over a window of nine residues. They are graphed with hydrophilic residues positive. Stretches with membrane-spanning potential consisting of about 20 mostly uncharged and hydrophobic residues with average hydrophobicities of at least 1.22 (16) are underlined. Their average hydropathies were calculated as follows: segment a (residues 262 to 280), +1.28; segment b (residues 713 to 732), +1.22; segment c (residues 294 to 323), +1.39; segment d (residues 373 to 397), +1.51; segment e (residues 311 to 332), + 1.52.

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TABLE 1. Features deduced from the predicted amino acid sequences of E. coli peptidases Peptidase

D N A P

Length Mol wt (amino acids) acids)

485 871 503 441

52,890 98,929 54,886 49,821

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Similarity of peptidase D to unrelated proteins. Stirling et al. (34) recently demonstrated the identity of the E. coli genes xerB and pepA. They suggested that peptidase A could play a functional role as an accessory factor in site-specific recombination of the ColEl plasmid. The similarity of regions of peptidase D to structural proteins like a viral matrix protein and human keratin may also be interpreted in this respect. Peptidase D, in addition to its enzymatic activity, may also function as a structural component in other cellular processes. ACKNOWLEDGMENTS The skillful and friendly assistance of Ulrike Theisinger and her personal interest are gratefully acknowledged. We thank Rainer W. Frank for the amino-terminal sequencing of peptidase D fragments and Matthias Redenbach for the synthesis of an oligonucleotide primer.

LITERATURE CITED 1. Baily, M., M. Foglino, M. Bruschi, M. Murgier, and A. Lazdunski. 1986. Nucleotide sequence of the promoter and aminoterminal encoding region of the Escherichia coli pepN gene. Eur. J. Biochem. 155:565-569. 2. Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of Si endonucleasedigested hybrids. Cell 12:721-732. 3. Burke, J. F. 1984. High-sensitivity S1 mapping with singlestranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene 30:63-68. 4. Doolittle, R. F., D. F. Feng, M. S. Johnson, and M. A. McClure. 1986. Relationships of human protein sequences to those of other organisms. Cold Spring Harbor Symp. Quant. Biol. 51: 447-455. 5. Foglino, M., S. Gharbi, and A. Lazdunski. 1986. Nucleotide sequence of the pepN gene encoding aminopeptidase N of Escherichia coli. Gene 49:303-309. 6. Gausepohl, H., M. Trosin, and R. Frank. 1986. An improved gas phase sequenator including on-line identification of PTH amino acids, p. 149-160. In B. Wittmann-Liebold, J. Salnikow, and V. A. Erdmann (ed.), Advanced methods in protein microsequence analysis. Springer-Verlag, New York. 7. Hartmann, R. K., and V. A. Erdmann. 1989. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit. J. Bacteriol. 171:2933-2941. 8. Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255. 9. Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351-359. 10. Henrich, B., U. Schroeder, R. W. Frank, and R. Plapp. 1989. Accurate mapping of the Escherichia coli pepD gene by sequence analysis of its 5' flanking region. Mol. Gen. Genet. 215:369-373.

11. Ikemura, T. 1981. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J. Mol. Biol. 151:389-409. 12. Ikemura, T., and J. E. Dahlberg. 1973. Small ribonucleic acids of Escherichia coli. I. Characterization by polyacrylamide gel electrophoresis and fingerprint analysis. J. Biol. Chem. 248:

5024-5032. 13. Kingston, R. E. 1987. Primer extension, p. 4.8.1-4.8.3. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 14. Klein, J., B. Henrich, and R. Plapp. 1986. Cloning and expression of the pepD gene of Escherichia coli. J. Gen. Microbiol. 132:2337-2343. 15. Konigsberg, W., and G. N. Godson. 1983. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc. Natl. Acad. Sci. USA 80:687-691. 16. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 17. Lazdunski, A. M. 1989. Peptidases and proteases of Escherichia coli and Salmonella typhimurium. FEMS Microbiol. Rev. 63: 265-276. 18. Makino, K., H. Shinagawa, M. Amemura, and A. Nakata. 1986. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J. Mol. Biol. 190:37-44. 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Messing, J., R. Crea, and P. H. Seeburg. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309-321. 21. Miller, C. G. 1987. Protein degradation and proteolytic modification, p. 680-691. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C. 22. Miller, C. G., and G. Schwartz. 1978. Peptidase-deficient mutants of Escherichia coli. J. Bacteriol. 135:603-611. 23. Murgier, M., and S. Gharbi. 1982. Fusion of the lac genes to the promoter for the aminopeptidase N gene of Escherichia coli. Mol. Gen. Genet. 187:316-319. 24. Norrander, J., T. Kempe, and J. Messing. 1983. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101-106. 25. Nuesch, J., and D. Schumperli. 1984. Structural and functional organization of the gpt gene of Escherichia coli. Gene 32: 243-249. 26. Richardson, K. K., J. Fostel, and T. R. Skopek. 1983. Nucleotide sequence of the xanthine guanine phosphoribosyl transferase gene of E. coli. Nucleic Acids Res. 11:8809-8816. 27. Rosenberg, M., and D. Court. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13:319-353. 28. Sawers, G., and A. Bock. 1989. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulator sequences and multiple promoters regulate anaerobic expression. J. Bacteriol. 171:2485-2498. 29. Selden, R. F. 1987. Analysis of RNA by Northern hybridization, p. 4.9.1-4.9.8. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 30. Sharp, P. M., and W.-H. Li. 1986. Codon usage in regulatory genes in Escherichia coli does not reflect selection for 'rare' codons. Nucleic Acids Res. 14:7737-7749. 31. Shinagawa, H., K. Makino, and A. Nakata. 1983. Regulation of the pho regulon of Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB. J. Mol. Biol. 168:477-488.

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32. Shine, J., and L. Dalgarno. 1974. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71:1342-1346. 33. Simmonds, S., and J. S. Fruton. 1949. The utilization of amino acids and peptides by mutant strains of Escherichia coli. J. Biol. Chem. 180:635-640. 34. Stirling, C. J., S. D. Coiloms, J. F. Collins, G. Szatmari, and D. J. Sheratt. 1989. xerB, an Escherichia coli gene required for plasmid ColEl site-specific recombination, is identical to pepA encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J. 8:1623-1627. 35. Stuber, D., I. Ibrahimi, D. Cutler, B. Dobberstein, and H. Bujard. 1984. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J. 3:3143-3148. 36. Wallace, R. B., and C. G. Miyada. 1987. Oligonucleotide probes

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