Inactivation of Cytochrome o Ubiquinol Oxidase Relieves Catabolic

JOURNAL OF BACTERIOLOGY, July 2002, p. 3785–3793 Vol. 184, No. 140021-9193/02/$04.00�0 DOI: 10.1128/JB.184.14.3785–3793.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inactivation of Cytochrome o Ubiquinol Oxidase Relieves CatabolicRepression of the Pseudomonas putida GPo1 Alkane

Degradation PathwayM. Alejandro Dinamarca, Ana Ruiz-Manzano, and Fernando Rojo*

Departamento de Biotecnologıa Microbiana, Centro Nacional de Biotecnologıa, CSIC, Campus dela Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 22 March 2002/Accepted 24 April 2002

Expression of the alkane degradation pathway encoded by the OCT plasmid of Pseudomonas putida GPo1 isregulated by two control systems. One relies on the transcriptional regulator AlkS, which activates expressionof the pathway in the presence of alkanes. The other, which is a dominant global regulation control, repressesthe expression of the pathway genes when a preferred carbon source is present in the growth medium inaddition to alkanes. This catabolite repression control occurs through a poorly characterized mechanism thatultimately regulates transcription from the two AlkS-activated promoters of the pathway. To identify thefactors involved, a screening method was developed to isolate mutants without this control. Several isolateswere obtained, all of which contained mutations that mapped to genes encoding cytochrome o ubiquinoloxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions. Elimina-tion of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertanimedium and in a defined medium containing lactate or succinate as the carbon source. This suggests thatcatabolic repression could monitor the physiological or metabolic status by using information from the electrontransport chain or from the redox state of the cell. Since inactivation of the crc gene also reduces catabolicrepression in rich medium (although not that observed in a defined medium), a strain was generated lackingboth the Crc function and the cytochrome o terminal oxidase. The two mutations had an additive effect inrelieving catabolic repression in rich medium. This suggests that crc and cyo belong to different regulationpathways, both contributing to catabolic repression.

The expression of bacterial catabolic pathways for the as-similation of aliphatic and aromatic hydrocarbons is frequentlysubject to complex control which links induction of the corre-sponding genes not only to the presence of the substrates to bedegraded but also to the proper physiological status of the cell(11). This is the case of the alkane degradation pathway en-coded by the OCT plasmid of Pseudomonas putida GPo1 (pre-viously named Pseudomonas oleovorans GPo1; see reference53). The genes of this pathway are grouped into two clusters,alkBFGHJKL and alkST (Fig. 1) (53, 54). The alkBFGHJKLoperon is transcribed from a promoter, PalkB, whose expres-sion requires the transcriptional activator AlkS and the pres-ence of alkanes (31, 42). In the absence of alkanes, the alkSTgenes are expressed from promoter PalkS1, which is recog-nized by �S-RNA polymerase (7, 8). Therefore, during expo-nential growth with a carbon source other than alkanes, thegene for the AlkS regulator is essentially silent. Upon entryinto the stationary phase, when �S-RNA polymerase becomesavailable, transcription of alkS from this promoter increases.AlkS acts as a repressor of PalkS1, allowing for low expressionof the alkST genes. When alkanes are present, the AlkS pro-tein bound to PalkS1 activates the expression of promoterPalkS2, located 38 nucleotides (nt) downstream from PalkS1,and provides for high expression of the alkST genes (7). There-

fore, the pathway is controlled by a positive feedback mecha-nism governed by AlkS (Fig. 1). The amount of AlkS protein ininduced cells is, however, low since this protein seems to bevery unstable (56, 57). The pathway is regulated by an addi-tional control system, since the levels of the alkane degradationenzymes are modulated by catabolic repression depending onthe carbon source being used (24, 47). This superimposedcontrol occurs through a poorly characterized mechanism thatultimately regulates transcription from promoters PalkB andPalkS2 (7, 56, 57). Activation of these promoters by AlkS andthe alkane inducer is very efficient when cells are grown in aminimal salts medium containing citrate as carbon source butshows a three- to fourfold reduction when organic acids such aslactate, pyruvate, or succinate are used as the carbon source.Repression is much stronger (ca. 50-fold repression) when cellsgrow exponentially in a rich medium such as Luria-Bertani(LB) medium or in minimal salts medium supplemented withCasamino Acids. Repression in rich medium abruptly disap-pears as cells enter the stationary phase of growth, whichsuggests the existence of elements that assure low-level expres-sion of promoters PalkB and PalkS2 during exponentialgrowth. The P. putida Crc protein plays an important role inthe catabolic repression observed in rich medium, although notthe one observed when cells grow in minimal salts mediumcontaining lactate or succinate as a carbon source (57). Inac-tivation of the crc gene reduces catabolic repression in richmedium by about sixfold, although a fivefold repression stilloccurs in conditions of no catabolic repression, indicating thatother elements besides Crc participate in this control (57). To

* Corresponding author. Mailing address: Centro Nacional de Bio-tecnologıa, CSIC, Campus de la Universidad Autonoma de Madrid,Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91-585-45-39. Fax:(34) 91-585-45-06. E-mail: [email protected].

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identify additional factors involved in the modulation of thispathway, a screening method was developed to obtain mutantsshowing a reduced catabolic repression in rich medium. Ninemutants were isolated. In all cases, mutations mapped to genesencoding the cytochrome o ubiquinol oxidase, the main termi-nal oxidase of the electron transport chain under highly aero-bic conditions. The aerobic respiratory chains of both Esche-richia coli and P. putida include a number of membrane-bounddehydrogenases that transfer electrons to ubiquinone, reduc-ing it to ubiquinol. This can then be oxidized by either of tworespiratory ubiquinol oxidases: the cytochrome o complex orthe cytochrome d complex (22). When cells grow exponentiallywith an ample supply of oxygen, cytochrome o oxidase (cyo)accommodates most of the electron flow. As the oxygen supplybecomes limiting, cytochrome d oxidase (cyd) is synthesized asan alternative terminal oxidase (15, 16, 49). Strains that lackeither cyo or cyd are fully capable of aerobic growth undernormal laboratory conditions, since the function of the missingoxidase seems to be taken over by the one remaining (1). Ananalysis was made of the influence of cytochrome o ubiquinoloxidase in the AlkS-mediated activation of the PalkB andPalkS2 promoters. The results suggest that catabolic repressionof the P. putida GPo1 alkane degradation pathway is linked tothe activity of the electron transport chain and/or to the redoxstate of the cell.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. The strains and plasmidsused are listed in Table 1. P. putida strains were grown at 30°C in LB medium orin M9 minimal salts medium (45), the latter supplemented with trace elements(4), and 30 mM citrate, lactate, or succinate as the carbon source. Expression ofthe PalkB or PalkS2 promoters was induced was demontrated by the addition of0.05% (vol/vol) dicyclopropylketone (DCPK), a nonmetabolizable inducer thatmimics the effect of alkanes (24). E. coli cells were grown in LB medium at 37°C.Antibiotics were added when appropriate at the following concentrations: am-

picillin, 100 �g/ml; kanamycin, 50 �g/ml; streptomycin, 50 �g/ml; and tetracy-cline, 8 �g/ml.

P. putida RC12 is derived from P. putida KT2442 by insertion of a PalkB::kantranscriptional fusion into its chromosome by using the suicide donor plasmidpALKRC1. This plasmid was generated as follows. A transcriptional fusion ofthe PalkB promoter to a kanamycin resistance (Kmr) determinant was generatedby excising a DNA fragment containing the PalkB promoter (positions �525 to�66 relative to the transcriptional start site) from plasmid pPB7 with KpnI(blunted with T4 DNA polymerase and deoxynucleoside triphosphates [dNTPs])and HindIII and inserting it between the HincII and HindIII sites of plasmidpUCKm. The plasmid obtained was named pPBK10. The PalkB::kan fusion wasexcised from pPBK10 with KpnI (blunted with T4 DNA polymerase and dNTPs)and EcoRI and inserted between the HindIII (blunted with T4 DNA polymeraseand dNTPs) and EcoRI sites of plasmid pUJ8. The plasmid obtained was namedpPBKJ1. The PalkB::kan fusion was excised from pPBKJ1 as a NotI DNAfragment and inserted at the NotI site of the mini-Tn5 suicide donor plasmidpJMT6, generating plasmid pALKRC1. P. putida RC12S8 is derived from strainRC12 by insertion of the alkS gene into its chromosome by using the suicidedelivery plasmid pTCS1. This plasmid is derived from mini-Tn5Tc by insertion atits NotI site of a NotI DNA fragment containing the alkS gene, which wasobtained from plasmid pUJS1.

To generate the �cyoB P. putida strain PBS4B1, a 1,200-bp DNA fragmentcontaining the cyoB gene was PCR amplified from P. putida KT2442 with theprimers 5�-CGGGATCCACGAAGAAGCAGGCAGCA-3� and 5�-CGGGATCCAGAACCAGAAGGCAG-3�, which contain BamHI sites at their 5� ends. Theproduct was digested with BamHI and cloned at the BamHI site of plasmidpUC18, yielding plasmid pCYOB. A 2-kbp EcoRI fragment containing a tetra-cycline resistance (Tcr) determinant was obtained from plasmid pUT-mini-Tn5Tc and inserted at the EcoRI site of pCYOB, located inside the cyoB gene.The plasmid generated was named pCYOBTc. Finally, a 3.2-kb BamHI fragmentfrom pCYOBTc containing the cyoB::tet allele was cloned into plasmidpKNG101, yielding pKCYOBTc. Plasmid pKNG101 is designed for markerexchange mutagenesis; it replicates in E. coli but not in P. putida and carries astreptomycin resistance (Smr) determinant and the sacB gene, which mediatessucrose sensitivity (29). Plasmid pKCYOBTc was transferred to P. putida PBS4in triparental matings with plasmid pRK600 as donor of transfer functions, andSmr Tcr sucrose-sensitive (8% [wt/vol]) exconjugants were selected. These cellswere expected to contain plasmid pKCYOBTc integrated into the chromosomeby a single recombination event at the cyoB gene, which generates a mutant anda wild-type cyoB gene. Cells having undergone a second recombination eventleading to loss of the wild-type cyoB allele were selected by screening for Tcr,streptomycin-sensitive (Sms), and sucrose-resistant colonies. The absence of thewild-type cyoB gene and the presence of the cyoB::tet allele were confirmed byPCR.

Inactivation of the crc gene in strain PBS4B1 was performed by markerexchange mutagenesis. The promoterless xylE reporter gene was excised fromplasmid pXYLE10 with endonuclease SmaI and inserted at the NruI site of thecrc gene present in plasmid pCRC5. The plasmid obtained was named pCRC5A.The crc::xylE allele was excised from pCRC5A by partial restriction with BamHIand cloned at the BamHI site of plasmid pKNG101, yielding plasmid pCRC20.This plasmid was transferred to strain PBS4B1 and exconjugants in which thewild-type crc gene had been substituted by the crc::xylE allele were isolated asdescribed above. A representative isolate was selected and named PBS4BC1.

Generation of mutant P. putida strains showing reduced catabolic repressionin LB medium. P. putida RC12S8 (KT2442 with a PalkB::kan fusion and alkS inthe chromosome) was mutagenized with minitransposon mini-Tn5Sm. This wasdelivered to the recipient cells in triparental matings as previously described (18)by using E. coli CC118�pir containing the plasmid pUT-mini-Tn5Sm as a donorand E. coli HB101 containing the plasmid pRK600 as a helper for transferfunctions. The exconjugants were plated on LB plates containing streptomycin,kanamycin, and 0.05% (vol/vol) DCPK and then incubated at 30°C for 24 to 48 h.Kmr colonies were streaked onto LB plates with kanamycin in the absence orpresence of DCPK. Colonies resistant to kanamycin in the absence of DCPKwere discarded (ca. 20%). Those selected were further tested for DCPK-depen-dent resistnace to kanamycin in LB broth. To identify the insertion point of theminitransposon, two approaches were used. For the mutant strains RCM4,RCM18, RCM2A, RCM3A, and RCM137A, chromosomal DNA was purifiedand digested with either KpnI (strains RCM4 and RCM18), SacI (strainsRCM2A and RCM3A), or KpnI�PstI (strain RCM137A), none of which cutinside the minitransposon. The digested fragments were ligated to pUC18 ex-cised with the same endonucleases and transformed into E. coli TG1. PlasmidDNA was extracted from Smr ampicillin-resistant (Apr) colonies, and the DNAinsert was sequenced in both strands. In all other mutant strains analyzed, the

FIG. 1. P. putida GPo1 alkane degradation pathway. The genes aregrouped into two clusters, alkBFGHJKL and alkST, both of which areregulated by the AlkS protein. In the absence of alkanes, AlkS isexpressed from promoter PalkS1; AlkS acts as a repressor of thispromoter, allowing for low expression levels. This promoter is recog-nized by �S-RNA polymerase, being active only in the stationary phaseof growth. In the presence of alkanes, AlkS activates expression fromthe PalkB and PalkS2 promoters, generating a positive amplificationloop on alkS expression. Activation of these two promoters by alkanesis strongly repressed by catabolic repression when cells grow exponen-tially in rich LB medium. Growth in a minimal medium containingsome organic acids (lactate or succinate) as a carbon source generatesa milder catabolic repression effect.

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point of insertion was identified by arbitrary PCR (6, 41) and was later verifiedby PCR with specific primers.

Assay for �-galactosidase. An overnight culture of the appropriate strain wasdiluted to a final turbidity (A 600) of ca. 0.04 in fresh LB medium, or in minimalsalts M9 medium supplemented with the indicated carbon source. When turbid-ity reached to 0.08, the nonmetabolizable inducer DCPK (0.05% [vol/vol]) wasadded where indicated. Cultures were grown at 30°C. At different time points,aliquots were taken and �-galactosidase activity measured as described by Miller(33). At least three independent assays were performed in each case.

S1 nuclease protection assays. Total RNA was isolated from bacterial culturesas previously described (34). S1 nuclease reactions were also performed aspreviously described (3) with 50 �g of total RNA and an excess of a 5�-end-labeled single-stranded DNA (ssDNA) hybridizing to the 5� region of themRNA. This allows detection of the transcription start sites, as well as detectionof the amounts of transcript generated. The ssDNA probe was obtained by linearPCR as described previously (56) by using either plasmid pTS1 linearized withHindIII (contains alkS and its promoter region) or plasmid pPB7 linearized withPstI (contains the PalkB promoter) as a substrate. The primers used hybridized

70 nt downstream of promoter PalkS2 or 73 nt downstream of promoter PalkB(the start sites were as described elsewhere [7]).

RESULTS

Generation of mutant strains showing reduced catabolicrepression in rich LB medium. To obtain mutant strains show-ing a reduced catabolic repression on the PalkB promoter inLB medium, strain RC12S8 was constructed (see Materialsand Methods). This strain, which derives from P. putidaKT2442, contains in its chromosome a copy of the alkS geneand a PalkB::kan transcriptional fusion in which a Kmr deter-minant is expressed from the AlkS-dependent PalkB promoter.Strain RC12S8 was sensitive to kanamycin when grown in theabsence of a positive effector of the AlkS regulator. In the

TABLE 1. Strains and plasmids

Strain or plasmid Description or relevant phenotypea Source or reference

StrainsE. coli

CC118(�pir) CC118 lysogenized with �pir phage 25HB101 Host for plasmid pRK600 5TG1 Host for DNA manipulations 45

P. putidaKT2442 hsdR Rif 21PBS4 KT2442 with PalkB::lacZ and alkS in the chromosome 56PBS4B1 PBS4 with an inactivated cyoB::tet allele This workPBS4C1 PBS4 with an inactivated crc::tet allele 57PBS4BC1 PBS4 with an inactivated crc::xylE and cyoB::tet alleles This workRC12 KT2442 with PalkB::kan in the chromosome This workRC12S8 KT2442 with PalkB::kan and alkS in the chromosome This workRCM4 RC12S8 with a mini-Tn5Sm insertion at the cyoC gene This workRCM18 RC12S8 with a mini-Tn5Sm insertion at the cyoB gene This workRCM2A RC12S8 with a mini-Tn5Sm insertion at the cyoB gene This workRCM3A RC12S8 with a mini-Tn5Sm insertion at ORF2 of the cyo cluster This workRCM6A RC12S8 with a mini-Tn5Sm insertion at the cyoA gene This workRCM22A RC12S8 with a mini-Tn5Sm insertion at the cyoE gene This workRCM117A RC12S8 with a mini-Tn5Sm insertion at the cyoB gene This workRCM137A RC12S8 with a mini-Tn5Sm insertion at the cyoA gene This workRCM2B RC12S8 with a mini-Tn5Sm insertion at the cyoE gene This work

PlasmidspALKRC1 Apr Telr, PalkB::kan transcriptional fusion into pJMT6 This workpCRC5 Apr; P. putida KT2442 crc gene cloned into pUC18 57pCRC5A Apr; crc::xylE allele into pUC18; derives from pCRC5 This workpCRC20 Smr; crc::xylE allele inserted at the BamHI site of pKNG101 This workpCYOB P. putida KT2442 cyoB gene cloned into pUC18 This workpCYOBTc cyoB::tet allele into pUC18 This workpJMT6 Apr Telr; mini-Tn5 suicide donor plasmid 46pKCYOBTc cyoB::tet allele into pKNG101 This workpKNG101 Smr; suicide vector for marker exchange mutagenesis 29pPB7 Apr; PalkB promoter into pUC19 56pPBK10 Apr; PalkB::kan fusion into pUCKm This workpPBKJ1 Apr; PalkB::kan fusion into pUJ8 This workpRK600 Cmr Mob� Tra�; donor of transfer functions 30pTCS1 Apr Tcr; alkS in mini-Tn5Tc This workpTS1 Apr; alkS into pT7-12 56pUC18 Apr; cloning vector 55pUCKm Apr; contains a promoterless Kmr cassette 13pUJS1 Apr; alkS gene cloned into pUJ8 56pUJ8 Apr; vector for transcriptional fusions to lacZ 18pUT-mini-Tn5Sm Apr Smr; mini-Tn5 suicide donor 18pUT-mini-Tn5Tc Apr Tcr; mini-Tn5 suicide donor 18pXYLE10 Kmr; P. putida xylE gene as an excisable cassette 48

a Telr, tellurite resistant; Rifr, rifampin resistant.

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presence of such an effector (an alkane, or the nonmetaboliz-able analogue DCPK), strain RC12S8 was resistant to kana-mycin when grown in a medium generating no catabolic re-pression (e.g., minimal salts medium containing citrate ascarbon source). However, this strain was sensitive to kanamy-cin when grown in LB medium in the presence of the inducerDCPK, as might be expected owing to the strong catabolicrepression that the LB medium imposes on PalkB induction.To search for mutants showing a reduced repression effect,strain RC12S8 was mutagenized with minitransposon mini-Tn5Sm. Nine mutant strains were isolated that were resistantto kanamycin in LB medium in the presence of inducer but notin its absence (see Materials and Methods). They were namedRCM4, RCM18, RCM2A, RCM2B, RCM3A, RCM6A,RCM22A, RCM117A, and RCM137A. The sequence of thePalkB and PalkS promoters in all of these strains was found tobe identical to that of the parental strain.

The point of insertion of the mini-Tn5Sm in these strainswas localized either by cloning and sequencing of the chromo-somal DNA segment containing the Smr determinant or byarbitrary PCR (see Materials and Methods). In mutant strainRCM4, the minitransposon was found inserted at an openreading frame showing 88% amino acid similarity to the cyoCgene of P. putida IH-2000 (28), which codes for subunit III(also known as subunit C) of cytochrome o ubiquinol oxidase(Fig. 2). In mutant strains RCM18, RCM2A, and RCM117A,the minitransposon was found to interrupt an open readingframe showing 96% amino acid similarity to the cyoB gene ofP. putida IH-2000 encoding subunit I (or subunit B) of thesame cytochrome (28). These two genes form part of the cyo-ABCDE cluster, which encodes subunits II, I, III, and IV of theP. putida cytochrome o oxidase complex (also known as the cyocomplex) and the heme o synthase (28, 36) (see Fig. 2). Cyto-chrome o ubiquinol oxidase is one of the two terminal ubiqui-nol oxidases in the P. putida respiratory chain (36). In the othermutant strains analyzed, the minitransposon was found to map

to other genes of the cyo cluster (Fig. 2), namely, the cyoA gene(strains RCM6A and RCM137A), and the cyoE gene (strainsRCM22A and RCM2B), or at ORF2 (strain RCM3A).

Expression of the PalkB and PalkS promoters upon inacti-vation of the cyoB gene. To ascertain that the reduction ofcatabolic repression observed in the above mutant strains wasdue to the inactivation of cytochrome o ubiquinol oxidaserather than to another unknown mutation and to have a reli-able system to measure the effect of this terminal oxidase oncatabolic repression, a knockout mutation was performed atthe cyoB gene of P. putida PBS4. This strain contains aPalkB::lacZ transcriptional fusion and the alkS gene insertedinto the chromosome and was the host used in previous studiesto analyze the catabolic repression of the PalkB promoter.Extensive work has shown that �-galactosidase levels in strainPBS4 faithfully reproduce the transcriptional activity of thePalkB promoter (56). The PBS4 cyoB gene was inactivated bymarker exchange mutagenesis by using a plasmid containing acyoB gene interrupted by a Tcr determinant (the cyoB::tet al-lele; see Materials and Methods); the strain obtained wasnamed PBS4B1. This strain showed normal growth in richmedium under the conditions used above and had essentiallythe same growth rate as the wild type. At least in E. coli, cellscontaining mutations in the cyo complex grow aerobically usingthe alternative cyd complex (2), which probably explains thenormal growth of strain PBS4B1. When grown in defined me-dium with citrate, a compound that does not generate catabolicrepression (56), activation of the PalkB promoter after theaddition of the inducer was equally efficient in strains PBS4and PBS4B1 (Fig. 3A). However, when cells were grown in LBmedium, PalkB induction was different in the two strains (seeFig. 3B). As previously described (56), activation of PalkB inthe wild-type strain PBS4 was very low during the exponentialphase of growth and strongly increased at the onset of thestationary phase. This occurred at turbidity values (A600) of ca.1.2 to 1.4. In the case of the cyo-deficient strain PBS4B1,

FIG. 2. Insertion points of mini-Tn5Sm in the P. putida catabolic repression mutants isolated. The upper scheme shows the genes encoding theP. putida cyo complex (28) and the positions of the mini-Tn5Sm insertions found in mutant strains RCM4, RCM18, RCM2A, RCM2B, RCM3A,RCM6A, RCM22A, RCM117A, and RCM137A (indicated by arrows). The cyoA, cyoB, cyoC, and cyoD genes encode subunits II, I, III, and IVof the oxidase complex, respectively; cyoE encodes the heme o synthase. The bottom scheme represents the electron transport chain. NDH, NADHdehydrogenase; LDH, lactate dehydrogenase; SDH, succinate dehydrogenase; UQ, oxidized ubiquinone; UQH2, reduced ubiquinone; cyo,cytochrome o ubiquinol oxidase.



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although PalkB activity was poor at the start of the exponentialphase, repression was clearly relieved as growth proceeded.The �-galactosidase levels increased almost fourfold at mid-exponential phase (A600 of 0.5) and more than ninefold at lateexponential phase (A600 of 1.0) compared to those with thewild-type strain PBS4 (Fig. 3B). If we consider the repressionvalues observed for the wild-type strain, inactivation of cyto-chrome o ubiquinol oxidase therefore reduced the catabolicrepression exerted by the LB medium by 3.4-fold at themid-exponential phase and by 7.5-fold at the late exponentialphase of growth (Table 2). Since repression in the wild type isin the range of 50- to 70-fold (Table 2), it is clear that inacti-vation of cytochrome o ubiquinol oxidase only provides partialrelief of the catabolic repression observed in LB medium.

The influence of the cyoB::tet mutation on the catabolicrepression observed in cells growing in a minimal salts mediumcontaining organic acids as the carbon source was also ana-lyzed. In the case of the wild-type strain, the use of succinate or

FIG. 3. Effect of the cyoB mutation on induction of the PalkB promoter in cells growing with different carbon sources. P. putida strains PBS4and PBS4B1 (PBS4 with a knockout mutation at the cyoB gene) were grown in duplicate flasks either in LB medium (B) or in minimal salts mediumcontaining either citrate (Cit) (A), succinate (Scc) (C), or lactate (Lac) (D) as the carbon source. At an A600 of 0.08, the nonmetabolizable inducerDCPK was added to one of the flasks; the other was left as a noninduced control. Aliquots were taken at different times, and the �-galactosidaseactivity was measured. The plots show the values observed for induced cultures (noninduced cultures had very low �-galactosidase activities [30to 100 Miller units] and are not represented). The values shown correspond to three to six independent assays, all of which are represented on thesame plot.

TABLE 2. Catabolic repression of the PalkB promoter in strainsPBS4 (wild type for cyoB) and PBS4B1 (cyoB::tet)

Medium

Activity (repression value)a in strain: Repressionreliefb

PBS4 PBS4B1

MEphase

LEphase

MEphase

LEphase

MEphase

LEphase

Citrate 2,500 (1) 4,800 (1) 2,800 (1) 6,000 (1) 1 1LB medium 50 (51) 70 (68) 190 (14.7) 670 (9) 3.4 7.5Lactate 450 (5.5) 1,350 (3.5) 1,000 (2.8) 3,100 (1.9) 2 1.8Succinate 750 (3.3) 3,000 (1.6) 3,900 (0.7) 6,000 (1) 3.3 1.6

a �-Galactosidase activities (in Miller units) in strains PBS4 and PBS4B1 at themid-exponential (ME; A600 of 0.5) or late-exponential (LE; A600 of 1) phase ofgrowth, growing in either LB medium or in a defined medium with citrate,lactate, or succinate as the carbon source, were obtained from Fig. 3. Repressionvalues (indicated in parentheses) were obtained by dividing the activity observedwhen cells used citrate as a carbon source by that observed when LB medium,lactate, or succinate was used.

b That is, the quotient between the repression observed in strain PBS4 and thatobserved in the cyo-deficient strain PBS4B1.



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lactate as the carbon source reduced PalkB induction at mid-exponential phase by about threefold in the case of succinateand by fivefold in the case of lactate versus the values observedwhen citrate was the carbon source (Fig. 3C and D; see alsoTable 2). Repression declined as cells approached the station-ary phase of growth. The picture was different in the strainlacking a functional cytochrome o ubiquinol oxidase. In thiscase, succinate allowed an induction of the PalkB promoter asefficiently as citrate (Fig. 3C), showing that the cyoB::tet mu-tation had totally eliminated catabolic repression. When lac-tate was the carbon source, a reduction in repression was alsoevident, but the increase in PalkB expression was only of abouttwofold (Fig. 3D). Inactivation of cytochrome o, therefore,seems to affect not only the catabolic observed in LB mediumbut also the repression exerted by organic acids in a definedmedium.

It was previously shown that the promoters PalkB andPalkS2 are similarly regulated by the AlkS protein and aresubject to the same catabolic repression effect (7, 57). Toanalyze whether inactivation of cytochrome o also eliminatedcatabolic repression of the PalkS2 promoter, the activity of thispromoter in cells growing in LB medium in the presence of theinducer DCPK was analyzed by S1 nuclease protection assays.As a control, the activity of the PalkB promoter was analyzedin parallel. As shown in Fig. 4, the levels of the transcriptsoriginated at the PalkS2 and PalkB promoters at the late ex-ponential phase of growth were approximately sevenfoldhigher in strain PBS4B1 than in the wild-type strain PBS4. Thisis direct evidence that inactivation of cytochrome o ubiquinoloxidase diminishes catabolic repression at both the PalkS2 andthe PalkB promoters in cells growing in LB medium. It shouldbe noted that when cells grow exponentially in the presence ofDCPK, promoter PalkS1 is inactive and is not influenced bycatabolic repression (7).

Inactivation of the crc and cyoB genes has an additive effectin reducing catabolic repression. Inactivation of the crc genedecreases the catabolic repression of promoters PalkB andPalkS2 in rich LB medium (57). If crc and cyo form part ofindependent regulation pathways, their simultaneous inhibi-tion should have an additive effect. On the contrary, if they arecomponents of the same regulation pathway, their simulta-neous inhibition should cause a decrease in catabolic repres-sion not greater than that exerted by any of the two elementsalone. To distinguish between these two possibilities, a P.putida strain was constructed in which both the crc and cyoBgenes had been inactivated by marker exchange mutagenesis(strain PBS4BC1). As shown in Fig. 5, at mid-exponentialphase the �-galactosidase activity in the wild-type strain was of50 Miller units; inactivation of the crc gene (strain PBS4C1)increased this activity to 220 U, whereas inactivation of thecyoB gene (strain PBS4B1) increased the activity to 190 U.Simultaneous inactivation of the crc and cyoB genes (strainPBS4BC1) led to �-galactosidase activities of 400 Miller units,a value that is about twice that observed in strains PBS4C1 orPBS4B1. This indicates that the catabolic repression effectexerted by the Crc and cyo functions is additive. It is likely,therefore, that Crc and cyo modulate induction of the PalkBpromoter through different pathways.

DISCUSSION

The results presented here show that inactivation of cyto-chrome o ubiquinol oxidase reduces the catabolic repressionthat occurs at the PalkB and PalkS2 promoters when cells growexponentially in rich LB medium or in a defined medium withsuccinate or lactate as the carbon source. Repression reductionwas only partial, since a clear repression effect was still ob-served. Inactivation of cyo did not affect growth rate signifi-cantly under the conditions used. Therefore, the lesser cata-bolic repression cannot be attributed to a decrease in thegrowth rate. Further, it is known that growth rate per se doesnot explain the catabolic repression of the alkane degradationpathway (56). Finally, mutations in many genes could havecaused a decrease in growth rate, but the mutations obtained insubsequent screenings repeatedly mapped to the cyo cluster.

The observed link between a component of the electrontransport chain and catabolic repression opens new ways tounderstand this global regulation process. There are some in-teresting examples of regulatory systems that monitor the flowof electrons through the electron transport chain and which

FIG. 4. Effect of the cyoB mutation on expression of the PalkB andPalkS2 promoters. Strains PBS4 and PBS4B1 were grown in LB me-dium supplemented with the inducer DCPK (0.05% [vol/vol]). At anA600 of 0.8, cells were collected and the total RNA was obtained. Thelevels of mRNA originated at the PalkB and PalkS2 promoters weremeasured by S1 nuclease protection assays in the presence of a largeexcess of the probe. Promoter PalkS1 is inactive under these conditions(7) and gave no signal (results not shown). The cDNA resistant to S1nuclease was resolved in a denaturing polyacrylamide gel, in parallelwith a DNA size ladder obtained by chemical sequencing of thessDNA used as probe.



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use that information to regulate the activity of specific genesand integrate different metabolic activities. For example, inRhodobacter sphaeroides the information obtained at twopoints of the electron transport chain is used to regulate theexpression of photosynthesis genes (37, 38). A signal generatedat the cbb3 branch of the electron transport chain is transducedto a two-component activation system which directly regulatesgene expression. In addition, the redox state of the quinonepool (the ubiquinol/ubiquinone ratio) is monitored by a redox-active antirepressor protein, which determines the functionalstate of a transcriptional repressor. The redox state of qui-nones is also used to regulate the transition from aerobic toanaerobic metabolism in E. coli by means of the Arc two-component system (23). In this case, the oxidized form of thequinones serves as a specific signal for the ArcB sensor kinase,silencing it and impeding phosphorylation of the ArcA globaltranscriptional regulator. It has also been proposed that theAer protein responds to the cellular redox state to regulateaerotaxis, which guides cells to oxygen-rich environments (44).Therefore, it is clear that bacterial cells can monitor the activ-ity of the electron transport chain and use this information toregulate gene expression. The results presented in the presentstudy suggest that the flow of electrons through the electrontransport chain, the redox state of the cell, or the amounts ofcyo ubiquinol oxidase (the levels of which are known to dependon the oxygen tension [15, 16, 49]) could be one of the signalsthat P. putida uses to modulate catabolic repression of thealkane degradation pathway. It cannot be ascertained at

present whether the cyo terminal oxidase has a direct or indi-rect role in this signal transmission process.

Inactivation of cytochrome o ubiquinol oxidase reduced, butdid not eliminate, catabolic repression (except that generatedby succinate). This suggests that catabolic repression dependson more factors than just the electron transport chain. It hasrecently been reported that the Crc protein has an importantrole in the catabolic repression of the alkane degradation path-way (57). Inactivation of the crc gene substantially reduced(although did not entirely eliminate) catabolic repression inrich medium but had no effect on the catabolic repressionexerted by lactate or succinate in a defined medium. Crc ap-pears to be an element of a signal transmission pathway con-necting cell physiology to carbon metabolism, participating incatabolic repression of some sugar and amino acid pathways(14, 26, 27), as well as in cell adhesion (40). Simultaneousinactivation of the cyoB and crc genes generated a greaterdecrease in catabolic repression in rich medium than the indi-vidual inactivation of either crc or cyoB. This additive effectsuggests that Crc and cyo form part of different signal trans-duction pathways, both of them contributing to catabolic re-pression. In both cases, the signal leads to a decrease in theAlkS-mediated induction of the PalkB and PalkS2 promoters(57; the present study). The final consequence would be adecrease in the levels of the AlkS regulator, which appears tobe present in the cell in limiting amounts (57). Keeping AlkSlevels below those required for full induction of the pathwayallows the cell to downmodulate expression of the alkane deg-radation genes when cells grow exponentially in medium con-taining a preferred carbon source in addition to alkanes. Inagreement with this idea, overexpression of the alkS gene froma strong heterologous promoter totally eliminates catabolicrepression (57).

Many other pathways for the degradation of linear or aro-matic hydrocarbons are subject to physiological control (globalregulation) in Pseudomonas spp. (17, 19, 32, 35, 39, 51), andthe mechanisms involved often differ. Comparison of the fac-tors implicated in the physiological control of the pWW0 tol-uene degradation pathway and of the P. putida CF600 phenoldegradation pathway provides a remarkable example of howsimilar regulatory outcomes can be achieved by using differentstrategies (50). Both pathways are controlled by �54-dependentregulator-promoter pairs that are mechanistically and func-tionally similar. In the two cases, activation of the correspond-ing promoters is modulated by a dominant physiological con-trol that generates a similar final effect. The host factorsresponsible for this physiological control differ in the two path-ways (9, 12, 13, 50, 51, 52). The effect of the cyo terminaloxidase on the regulation of these pathways has not beenspecifically addressed. However, it is worth noting that thecatabolic repression generated by excess succinate on expres-sion of the pWW0 toluene degradation pathway decreasesconsiderably when O2 is limited (19, 20). In such conditions,the cyo terminal oxidase should decrease in favor of the cydoxidase. This suggests the cyo terminal oxidase could also havesome role in the repression of this pathway. Interestingly, thecatabolic repression exerted by succinate on the expression ofthe phenol degradation pathway encoded in the P. putida Hplasmid pPGH1 is reduced by inactivation of the cyo terminaloxidase (43). However, the effect of LB medium on the expres-

FIG. 5. Effect of the simultaneous inactivation of the crc and cyogenes on catabolic repression of the PalkB promoter. P. putida strainsPBS4, PBS4B1 (PBS4 with a knockout mutation at the cyoB gene),PBS4C1 (PBS4 with a knockout mutation at the crc gene), andPBS4BC1 (PBS4 with knockout mutations at the cyoB and crc genes)were grown in duplicate flasks in LB medium. At an A600 of 0.08, thenonmetabolizable inducer DCPK was added to one of the flasks,whereas the other flask was left as a noninduced control. Aliquots weretaken at different times, and the �-galactosidase activity was measured.The levels of �-galactosidase are represented as a function of cellgrowth. The plot shows the values observed for induced cultures (non-induced cultures had very low �-galactosidase activities [30 to 90 Millerunits] and are not represented). The values shown correspond toseveral independent assays, all represented on the same plot.



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sion of this pathway has not been not reported. The role of thecyo terminal oxidase on the expression of other pathways has,to our knowledge, not been analyzed. The available data donot allow a unified picture of catabolic repression in Pseudo-monas spp. to be drawn. However, the mechanisms used ineach case seem to rely heavily on factors dictated by the pro-moters and regulators of the catabolic pathway (e.g., sigmafactors involved in promoter recognition, regulator stability,etc.). It is likely that the physiological status of the cell isconnected to gene expression in more than one way. Althoughcertain mechanisms will probably be more suited to a particu-lar pathway (or promoter-regulator pair) than to others, theevolutionary history of each pathway may be important indetermining which factors participate in each case (10, 50).This could provide substantial diversity to physiological controlmechanisms. However, more knowledge on the mechanismsmediating global control of the catabolic pathways of Pseudo-monas sp. is required before final conclusions can be drawn.

ACKNOWLEDGMENTS

We are grateful to M. Marın for helpful discussions and to L. Yustefor technical assistance.

This work was supported by grants BIO2000-0939 from the Comi-sion Interministerial de Ciencia y Tecnologıa and 07 M/0120/2000from Comunidad Autonoma de Madrid. M.A.D. was the recipient offellowships from the Instituto de Cooperacion Iberoamericana and theComision Nacional de Investigacion Cientıfica y Tecnologica/BancoInteramericano de Desarrollo (Chile). A.R.-M. was the recipient of afellowship from Gobierno Vasco.

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Inactivation of Cytochrome o Ubiquinol Oxidase Relieves Catabolic