New insights into the regulation of the pac gene from Escherichia coli W ATCC 11105

New insights into the regulation of the pac gene fromEscherichia coli W ATCC 11105

Ana Roa, Jose Luis Garc|a *Department of Molecular Microbiology, Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cient|¢cas, Velazquez 144,

28006 Madrid, Spain

Received 25 March 1999; received in revised form 28 May 1999; accepted 2 June 1999

Abstract

The regulation of the pac gene encoding the penicillin G acylase of Escherichia coli W ATCC 11015 has been investigated bya molecular approach using lacZ as a reporter gene. This analysis revealed that a region of 170 bp located upstream of the pacstructural gene contains the regulatory sequences that control its expression. The cAMP receptor protein is involved not only inthe catabolite repression of penicillin G acylase production caused by glucose but also in the induction of pac gene expressionby phenylacetic acid. Primer extension analyses have demonstrated that the transcription of the pac gene can be initiated fromat least three different promoters. Although all these promoters are functional, their relative activity depends on the transcribedgene, the P1 and P3 promoters being more active in the presence of the pac gene, whereas the P2 promoter was stronger whenthe upstream region of the pac gene was fused to the lacZ reporter. A deletion of the region surrounding the 310 box of the P3promoter produced a constitutive expression of the fused gene indicating that this sequence is required for phenylacetic acidinduction and suggesting that the expression of the pac gene is regulated by a repression mechanism. This work reveals that theregulation of the pac gene is more complex than previously envisioned and provides new clues to investigate further thisinteresting regulatory system. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.All rights reserved.

Keywords: Phenylacetic acid; Penicillin G acylase; Pac regulation

1. Introduction

Penicillin G acylase (PGA, penicillin G amidohy-drolase, EC 3.5.1.11) catalyzes the hydrolysis of ben-zyl penicillin and it is one of the most importantindustrial enzymes since it is used for the commercial

production of semisynthetic penicillins [1]. Althoughdi¡erent PGAs have been described in many micro-organisms, the enzyme from Escherichia coli WATCC 11105 has been the most extensively studiedso far [1]. The expression of the pac gene is subjectedto several regulatory controls, including temperature,oxygen, catabolite repression, and induction by phe-nylacetic acid (PAA) [1,2]. In addition, a complexmaturation process is required to render the activeform of PGA [2]. The enzyme is produced as aninactive cytoplasmic precursor of 93 kDa which be-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 8 1 - 5

* Corresponding author. Tel. : +34 (91) 5611800. Fax:+34 (91) 5627518; E-mail: [email protected]

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FEMS Microbiology Letters 177 (1999) 7^14

comes catalytically active after its translocation tothe periplasmic space followed by an autoproteolyticdisruption into two subunits (K and L) that removesan internal spacer polypeptide [2]. Since the pac genehas been cloned and sequenced [3,4], very few studieshave been done on the characterization and regula-tion of the pac promoter [5^7]. Some results reportedin the available literature that are a matter of dis-crepancy (see below) and the current pending ques-tions about PAA induction and catabolite repressionprompted us to reevaluate the regulatory elementsinvolved in the expression of this important gene.

In the present work, we have analyzed the sequen-ces involved in the expression of the pac gene of E.coli W ATCC 11105 using a lacZ fusion approachthat has provided a new scenario to understand thecomplex regulation of this gene.

2. Materials and methods

2.1. Bacteria and plasmids

The strains and plasmids used in this work areshown in Table 1. E. coli cells were grown with shak-ing in Luria broth (LB) [13] or M63 minimal me-dium [14]. Media were supplemented with L-leucine(100 Wg ml31), L-proline (100 Wg ml31), ampicillin(100 Wg ml31), tetracycline (12.5 Wg ml31), glucoseor glycerol when required.

2.2. DNA and RNA manipulations

Plasmid DNA was prepared by the rapid alkalinemethod [13]. Transformation of E. coli cells was car-ried out using the RbCl method [13]. Polymerasechain reaction (PCR) was performed using theGene ATAQ Controller (Pharmacia LKB). DNAfragments were puri¢ed using L-agarase (New Eng-land Biolabs, Beverly, MA, USA). DNA sequencingwas carried using the Pharmacia T7 sequencing kit.RNA extraction and primer extension analysis wereperformed as previously described [12], using the oli-gonucleotides PRS551 (5P-GCCAGGGTTTTCC-CAGTC-3P) and PAC (5P-GCTCCAATAATACAT-CAGGGAAG-3P) that hybridized within the 5Pcoding sequences of the lacZ and pac genes, respec-tively. PCR ampli¢cation was performed using theplasmid pPGA1 as template and the oligonucleotidesPEC1 (5P-CCCAAGCTTTTCATTGTATCCTTCT-GG-3P ; the BamHI site is underlined) and PEC2(5P-CGCGGATCCAGCGGTGAATAAAGCG-3P ;the HindIII site is underlined) as primers.

2.3. Enzyme assays

L-Galactosidase (L-Gal) activity was determinedaccording to the method of Miller [14] using 2-nitro-phenyl-L-galactopyranoside as substrate. PGA activ-ity was assayed using 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) as substrate [12]. Usually

Table 1Bacterial strains and plasmids used in this study

Strain or plasmid Relevant genotype or properties Source

E. coliMC4100 v(lacIPOZYA)U169, thi M. CasadabanSBS688 MC4100 vcrp39 J. PerezHB101 proA2, leuB, thi CIB collectionW ATCC 11105 Vitamin B12 auxotroph ATCCPlasmidspSKS107 amp, promoter-less lacZYA [8]pRS550 amp, kan, promoter-less lacZYA [9]pPGA1 tet, Ppac-pac from E. coli W [10]pAJ19 amp, vPpac-pac from E. coli W [11]pSKSK amp, Ppac(K. citrophila)-lacZ [12].pSKSP amp, Ppac-pac from E. coli W This studypSKSE amp, Ppac(E. coli)-lacZ This studypRS55E amp, vPpac(E. coli)-lacZ This study

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three independent determinations were made foreach activity measured.

3. Results and discussion

3.1. Expression of the pac gene in E. coli K12 strains

To con¢rm that the expression of the cloned pacgene was still inducible by PAA as occurs in theparental E. coli W ATCC 11105 [10], we determinedthe PGA activity produced in an E. coli K12 re-combinant strain harboring the pac gene. Table 2shows that the production of PGA in E. coliHB101 (pPGA1) (Fig. 1B) was inducible by PAA,whereas E. coli HB101 (pAJ19) produced PGA con-stitutively. Sequencing of plasmid pAJ19 [11] re-vealed that the cloned fragment only contains the¢rst 53 bp of the 5P non-coding region of the pacgene that exclusively harbors the 310 box of thepreviously proposed pac promoter [6] (Fig. 1B, here-after named P3 promoter). Therefore, the expressionof the pac gene in the pAJ19 recombinants is morelikely directed by a promoter present in the plasmid.However, we should also consider that Oh et al. [4]have ascribed the pac promoter to the sequencesTTGCTA (335 box) and TATACA (310 box) lo-cated downstream of the HindIII site (Fig. 1A). In-terestingly, the PAA induction of pac expression canbe restored (Table 2) by reconstructing in plasmidpSKSP the complete native upstream regulatory re-gion using a 170-bp PCR ampli¢ed fragment (Fig.1B). Therefore, this result suggested that the 170-bpfragment contained the regulatory signals required tocontrol pac expression, opening the possibility of

studying the pac promoter by using a simpli¢ed ex-perimental approach based on the construction oflacZ fusions.

3.2. Construction of L-Gal fusions

Although a preliminary analysis of pac expressionby fusing its non-coding upstream region to the lacZgene had been performed [5], this study presentedsome technical drawbacks hampering the interpreta-tion of the results. In this sense, the promoter anal-ysis was conducted in E. coli HB101, a lacZ strainthat displays a L-Gal background activity. In addi-tion, the lacZ fusion was constructed using the avail-able restriction sites of the pac sequence whichcaused that the cloned region was too large and ren-dered a chimeric L-Gal fused to the PGA signal pep-tide that might cause toxic e¡ects [15]. To avoid allthese drawbacks, we have constructed plasmidpSKSE (Fig. 1B), which contains the ¢rst 170 bpof the 5P non-coding region of the pac gene fusedto the ATG codon of the lacZ gene. When this plas-mid was transformed into E. coli MC4100 (vlacZ),we observed that the production of L-Gal was indu-cible by PAA (Table 3) supporting our suggestionthat the PCR-ampli¢ed sequence contains the signalsrequired for PAA induction. In addition, this resultsuggests that the presence of the complete pac gene isnot essential for PAA induction, refuting the hypoth-esis that the regulatory gene responsible for suchinduction is located within the pac structural gene[7]. Nevertheless, we cannot discard that such regu-latory element could play another role (see below).The e¡ect of PAA seems to be mediated by a regu-latory mechanism that is also present in a heterolo-gous host like E. coli HB101, lacking the pac gene.Since we have recently shown that the genomes of E.coli W ATCC 11105 and E. coli K12 encode thepathway for PAA degradation [16], we cannot ruleout the possibility that the gene(s) involved in theregulation of this pathway could also be involvedin pac gene expression.

On the other hand, we have also observed that theexpression of the lacZ fusion in E. coli MC4100(pSKSE) decreased when glucose was added to themedium (Table 3). Interestingly, the inhibitory e¡ectof glucose was lower on the uninduced cells (basallevel) than on the cells induced by PAA. Neverthe-

Table 2PGA activity of E. coli HB101 cells harboring di¡erent pac-con-taining plasmids

Plasmid PGA activitya (nmol min31 ml31 of culture)

3PAA +PAA

pPGA1 10 þ 5 240 þ 10pAJ19 220 þ 15 270 þ 15pSKSP 20 þ 5 260 þ 20

aCells were grown for 24 h at 28³C in LB medium supplementedwith tetracycline (12.5 Wg ml31) (pPGA1) or ampicillin (100 Wgml31) (pSKSP and pAJ19) in the absence or presence of 0.1%PAA. PGA activities were determined using NIPAB as substrate.

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Fig. 1. Plasmid constructions and nucleotide sequence of the 5P non-coding region of the E. coli W ATCC 11105 pac gene. A: Sequenceof the 5P non-coding region of pac gene from E. coli W ATCC 11105. CRP1 and CRP2 indicate the putative CRP binding sites. The 335and 310 boxes of the P1, P2 and P3 promoters are underlined. ATG in bold face represents the translation initiation codon of the pacgene. RBS shows the position of the ribosome binding site. +1 indicates the transcription start sites of the promoters. B: Schematic repre-sentation of the plasmid constructions. White boxes represent the putative CRP binding sites. The 335 and 310 boxes of P1, P2 and P3promoters are represented by dashed boxes. The lacZ gene is shown by a dotted box. The pac gene is represented by a striped box. Theoligonucleotides used for the PCR ampli¢cation are indicated as primers PEC1 and PEC2. Abbreviations: amp, ampicillin resistancegene; tet, tetracycline resistance gene; kan, kanamycin resistance gene; B, BamHI; H, HindIII; S, SacI; Sa, SalI, Sm, SmaI. H* indicatesa partial digestion with HindIII. H(Kl.) indicates a HindIII digestion followed by a treatment with the Klenow fragment of DNA poly-merase.

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less, PAA was still able to induce the production ofL-Gal in the presence of glucose, in both LB andM63 minimal medium, but the induction e¡ect wassigni¢cantly reduced (Table 3). The L-Gal produc-tion can be repressed up to 95% in LB medium con-taining 10 mM glucose, whereas the presence of 20mM glycerol only produced a limited repression(25%) (data not shown). These results are in agree-ment with previous ¢ndings [5] and strongly suggestthat the pac promoter is regulated by catabolite re-pression that can be most probably ascribed to thepresence of two putative CRP (cAMP receptor pro-tein) binding sites, CRP1 and CRP2, which are lo-cated within the 170-bp 5P non-coding region of thepac gene cloned in pSKSE (Fig. 1A).

To determine the in£uence of the CRP protein onthe regulation of the pac promoter the expression ofthe lacZ fusion in pSKSE was analyzed in the E. coliCRP3 mutant SBS668. The production of L-Gal inE. coli SBS688 (pSKSE) cells induced with PAA wasreduced to the basal levels independently of the ab-sence or the presence of 5 mM glucose in the culturemedium (Table 3). As expected, the addition of 5 mMglucose to the culture medium did not generate aninhibitory response on the uninduced cells (Table 3).The levels of L-Gal in the uninduced cells of boththe wild-type and the CRP3 strains were quite sim-ilar and they were not drastically a¡ected by theaddition of glucose suggesting that the basal expres-sion of lacZ fusion is mainly generated from a CRP-independent transcription activity. Surprisingly,these results not only con¢rmed that the cataboliterepression of pac promoter was a CRP-dependentprocess but, more important, they revealed that theCRP protein was directly involved in the PAA in-duction.

3.3. Analysis of pac promoter

In spite of the high similarity of the pac genesfrom E. coli W ATCC 11105 and Kluyvera citrophila,the pac promoter of K. citrophila was located over-lapping the sequence of the CRP2 binding site [12],this is, far upstream from the promoter (P3 pro-moter) proposed for the pac gene of E. coli WATCC 11105 [6] (Fig. 1A). This ¢nding pointed tothe possibility that other promoters might be in-volved in the expression of the E. coli pac gene. Toinvestigate this possibility, we constructed plasmidpRS55E that contains a lacZ gene fused to a frag-ment lacking the 310 box of the previously proposedpac promoter [6] or P3 promoter (Fig. 1B). Interest-ingly, E. coli MC4100 (pRS55E) cells cultured in LBmedium showed a high L-Gal production (3000 Uafter 20 h of culture) that was not induced byPAA (data not shown), but that was repressed by5 mM glucose (1000 U after 20 h of culture) (datanot shown). These results strongly supported the ex-istence of a pac promoter upstream of the HindIIIsite, and also indicated that the deleted region wasinvolved in the PAA induction. In this sense, thehigh constitutive activity found in these cells suggeststhat the pac expression might be regulated by a re-pressor that recognizes a binding site within the de-leted region.

A primer extension analysis carried out in E. coliMC4100 (vlacZ) transformed with plasmid pSKSE,showed the presence of three bands of di¡erent in-tensities (Fig. 2). The same pattern of bands wasobserved when the RNA was extracted after 20 h(Fig. 2, lane E1) and 3 h (Fig. 2, lane E3) of incu-bation, suggesting that the relative amounts and thelength of the transcripts were not a¡ected by the

Table 3L-Gal activity of E. coli MC4100 and E. coli SBS688 pSKSE transformants

Strain L-Gal activity (units)a

LB LB+5 mM glucose M63+5 mM glucose

3PAA +PAA 3PAA +PAA 3PAA +PAA

MC4100 (pSKSE) 330 þ 60 1500 þ 130 220 þ 60 430 þ 50 130 þ 40 720 þ 60SBS688 (pSKSE) 410 þ 50 400 þ 50 330 þ 60 400 þ 60 230 þ 50 230 þ 50

aActivities are expressed in Miller units. Cells were grown for 24 hours at 28³C in LB or M63 minimal medium supplemented with ampi-cillin (100 Wg/ml) in the absence or presence of 0.1% PAA and in the absence or presence of 5 mM glucose.

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phase of growth. The major band corresponded toa transcriptional start site located at 377 bp (P2promoter) from the ATG translation initiation co-don whereas the two minor bands corresponded toputative start sites located at a distance of 381 bp(P1 promoter) and 330 bp (P3 promoter), respec-tively (Figs. 1 and 3). Interestingly, the major bandcorresponded to that previously found in the trans-formants carrying the plasmid pSKSK containingthe lacZ gene fused to the pac promoter of K. citro-

phila (Fig. 3, lane E2) [12]. Upstream of the mainstart site we found the sequences TTAGTT andCATAAT that might represent the 335 and 310boxes of the P2 promoter (Fig. 1A). These sequencesoverlap the putative CRP2 binding site and do notperfectly match with the consensus sequence of theE. coli c70 promoters. The 335 box of P2 promoteris preceded by a putative A+T rich enhancer se-

Fig. 2. Primer extension analysis of the transcription start sitesof lacZ fusions. Total mRNA was isolated from cells cultured at28³C in LB medium containing ampicillin (100 Wg ml31) and0.1% PAA. Lane E1, mRNA isolated from E. coli MC4100(pSKSE) after 20 h of culture; lane E2, mRNA isolated from E.coli MC4100 (pSKSK) after 3 h of culture; lane E3, mRNA iso-lated from E. coli MC4100 (pSKSE) after 3 h of culture. Thesize of the extended products was determined by comparisonwith a DNA sequencing ladder of the pac promoter region usingthe plasmid pSKSE as template. The primer extensions and thesequencing reactions were performed using the primer PRS551.

Fig. 3. Primer extension analysis of the pac transcription startsites. Total mRNA was isolated from cells cultured at 28³C inLB medium containing tetracycline (12.5 Wg ml31) and 0.1%PAA. Lane E1, mRNA isolated from E. coli W ATCC 11105(pPGA1) cells after 6 h of incubation; lane E2, mRNA isolatedfrom E. coli HB101 (pPGA1) cells after 6 h of incubation; laneE3, mRNA isolated from E. coli HB101 (pPGA1) cells after 15 hof incubation. The size of the extended products was determinedby comparison with a DNA sequencing ladder of the pac pro-moter region using the plasmid pPGA1 as template. The primerextensions and the sequencing reactions were performed usingthe primer PAC.

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quence [17]. The start site located at 381 bp could beascribed to the 335 (TATCAA) and 310 (CAC-GAT) boxes of the putative P1 promoter. The exis-tence of a P1 and P2 promoters explains the expres-sion of lacZ observed with plasmid pRS55E. Finally,the putative start site located at 330 bp from theATG codon corresponds to that determined by Valleet al. [6] and, thus, the P3 promoter is built by the335 (TAGATA) and 310 (TAGTAT) boxes thatsurround the HindIII site (Fig. 1A).

It was surprising that the major transcription startsite did not correspond to the start site previouslylocated at 3-30 bp by Valle et al. [6]. To rule out thepossibility that the band of the P3 promoter mightbe generated by an unspeci¢c arrest of the reversetranscriptase or by a degradation/processing of pacmRNA, we reproduced the experiment carried outby Valle et al. [6]. Therefore, a primer extensionanalysis was performed using as hosts both the E.coli strains W ATCC 11105 and HB101 transformedwith plasmid pPGA1. Surprisingly, in this case, themain transcription start site was located at 330 bpfrom the ATG codon (Fig. 3). In contrast, the bandscorresponding to the P1 and P2 promoters were sig-ni¢cantly reduced compared to that of P3 promoter.The relative intensities and the positions of the bandswere similar when the RNA was extracted after 6 or15 h of culture (Fig. 3, lanes E2 and E3) indicatingthat length of the transcripts was independent of theincubation time. The same pattern was observed inboth W and HB101 strains suggesting that the dra-matic change in the intensities of the bands shouldnot be ascribed to the presence of speci¢c regulatoryelements in the parental strain.

The above ¢ndings strongly suggest that the pacgene a¡ects its own expression, modifying the tran-scription rate of the P1, P2 and P3 promoters. In thissense, it has been suggested that the pac gene mightcontrol its own expression [7], and although this ar-gument cannot be used to explain the PAA inductionobserved in plasmid pSKSE, we should consider thatthe PacR protein could modulate the a¤nity ofRNA polymerase for the alternative binding sites.Moreover, the upstream region of pac gene presentsseveral regions that could be involved in DNA bend-ing facilitating the interactions of di¡erent factors(CRP, PAA-dependent repressor, or PcaR) thatmight function as transcriptional switches between

tandem promoters [17]. Finally, it has been demon-strated that some genes such as the luxAB genes canactivate or repress transcription from a subset ofpromoters due to an intrinsically curved DNA seg-ment in the 5P coding sequence of the luxA gene [18].

Summarizing, the results presented in this workprovide novel experimental evidence that the regula-tion of the pac gene is more complex than previouslyenvisioned. The existence of several alternative pro-moters raises new questions about the nature of theregulatory elements and mechanisms that control theexpression of this gene that should be further inves-tigated.

Acknowledgements

We thank M.A. Prieto, E. D|az, and R. Lopez forcritical reading of the manuscript. The artwork of A.Hurtado and the technical assistance of E. Cano andM. Carrasco are gratefully acknowledged. This workwas supported by the Comision Interministerial deCiencia y Tecnolog|a (Grant AMB94-1038-CO2-02and Grant AMB97-603-CO2-02).

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New insights into the regulation of the pac gene from Escherichia coli W ATCC 11105