a novel multicopy suppressor of a groel mutation from different

The EMBO Journal vol.12 no.3 pp.889-896, 1993

A novel multicopy suppressor of a groEL mutationincludes two nested open reading frames transcribedfrom different promoters

Tsvika Greener, David Govezensky andAda Zamirl

Biochemistry Department, Weizmann Institute of Science, Rehovot76100, IsraellCorresponding author

Communicated by N.Sharon

When present on a multicopy plasmid, a newlydiscovered gene (sugE) mapping to 94 min on theEscherichia coli chromosome, suppresses a groELmutation and mimics the effects ofgroE overexpression.A groEL mutant of E.coli, transformed with theKiebsiella pneumoniae nif gene cluster, failed toaccumulate nitrogenase components [Govezensky et al.(1991) J. Bacteriol., 173, 6339-6346]. Transformationwith sugE reversed the mutant phenotype. In wild typeK.pneumoniae, transformation with sugE accelerated therate of nitrogenase biogenesis after nif derepression. InE.coli, transformation with sugE enabled bacteriophageT4 growth in a groEL mutant. A continuous 178 codonopen reading frame (ORF) in sugE encloses another, in-frame, 105 codon ORF similar to a predicted ORF inProteus vulgaris. In vivo products of both sugE ORFswere observed in transformants expressing the gene froma T7 promoter. In non-transformed cells, a typicalor70-dependent promoter found upstream of the largerORF directs sugE transcription during growth at 30°C.At elevated temperatures or in stationary phase cells,another promoter, found within the coding sequenceupstream of the smaller ORF, is activated independentlyof a32. The results suggest that sugE encodes achaperonin-related system whose composition might varywith temperature and growth phase.Key words: bacteriophage assembly/heat-shock/molecularchaperones/nitrogen fixation/stationary phase

IntroductionMolecular chaperones are proteins that assist the correctfolding of other proteins and thereby facilitate the formationof native protein conformations, protein oligomerization andtransport across membranes. Their interaction with fully or

partially denatured proteins also underlies the ability ofmolecular chaperones to counteract cellular damage by heat-shock and other types of stress. Molecular chaperones playa central role in a variety of cellular processes and, in mostinstances, are essential for cellular viability as well as growthat elevated temperatures (for reviews see Georgopoulos andAng, 1990; Ellis and van der Vies, 1991; Zeilstra-Ryallset al., 1991; Gething and Sambrook, 1992).The Escherichia coli GroEL chaperonin, a representative

of a universally conserved family of molecular chaperones,fulfils key functions in bacterial physiology as well as in

Oxford University Press

phage morphogenesis (Ellis and van der Vies, 1991; Zeilstra-Ryalls et al., 1991). GroEL and the auxiliary GroES areencoded in a single operon transcribed from two differentpromoters: one is a70-dependent and ensures constitutiveexpression and another depends on a32, the sigma factorrequired for transcription of the major heat-shock-inducedgenes in E. coli (Georgopoulos and Ang, 1990; Zeilstra-Ryalls et al., 1991).

Early genetic analyses placed mop (a synonym of groE)in two partially overlapping plasmids of the Clarke-CarbonE.coli gene library: pLC16-43 and pLC43-46, mapping to94 min on the E. coli chromosome (Neidhardt et al., 1983).However, more recent genetic and DNA sequencing analysesindicate that the groE operon maps to the part of pLC43-46not shared by pLC16-43 (Lohmeier et al., 1981; Guestet al., 1984; Fayet et al., 1986; Jenkins et al., 1986;Hemmingsen et al., 1988). The previous assignment of mopto pLC16-43 therefore suggested the possibility that thisplasmid might carry a novel gene(s) encoding a chaperonin-related function able to suppress groEL mutations. In viewof the profound significance of molecular chaperones ingeneral, and GroEL in particular, we have considered itworthwhile to pursue further the nature of this gene(s).The assays initially used were based on our studies of the

role of GroEL in bacterial nitrogen fixation (Govezenskyet al., 1991). In this process, reduction of atmosphericdinitrogen to ammonia is carried out by two oligomericmetalloproteins: the tetrameric (U2(32) MoFe protein and thedimeric (a2) Fe protein (Smith et al., 1988). In Klebsiellapnewnoniae, the information required for the process resideswithin a single nifgene cluster containing 20 genes. The nifgenes are arranged in 7-8 transcriptional units that forma regulon responding to the oxygen and fixed nitrogen statusof the cells via the synthesis and activity of NifA, the nif-specific transcriptional activator (Arnold et al., 1988;Merrick, 1988).Our previous analyses of foreign hosts expressing selected

K.pneumoniae nif genes have indicated a role for the hostorganism in the oligomerization of the two MoFe proteinstructural polypeptides (Berman et al., 1985a,b; Hollandet al., 1987). The possibility that the host function was thatof a molecular chaperone was supported by thedemonstration that GroEL transiently bound the nascentnitrogenase structural polypeptides and thereby probablyassisted their proper oligomerization. Most interestingly, thechaperonin was also found to be required for the fullactivation of nif promoters, a requirement most likelyemanating from the interaction of GroEL with NifA. Theregulatory role of GroEL was expressed most clearly in itseffect on the rate and level of accumulation of the nitrogenasecomponents (Govezensky et al., 1991).Based on the nifassays and subsequently on bacteriophage

T4-induced cell lysis, the present study identified amulticopy suppressor of a groEL mutation within thechromosomal fragment cloned in pLC16-43. The suppressor

889

T.Greener, D.Govezensky and A.Zamir

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Fig. 1. Effect of pDG1O on the accumulation of the MoFe protein in anE.coli groEL mutant. A. Ecoli strain T850 (groEL-) transformed withpWK220 (lanes 3,4) or co-transformed with pWK220 and pDG1O (lane 5),E.coli KL355 transformed with pWK220 (lane 2) and wild typeKpneunoniae (lane 1), were subjected to nif-derepressing conditions for8 h. Anaerobic protein extraction, non-denaturing gel electrophoresis andimmunoblotting with antibodies against MoFe protein from Kpneumoniae(Kpl) were as described in Materials and methods. An extract of derepressedwild type Kpneumoniae was run as a standard (lane 1). The bands markedas Kpl represent native MoFe protein and two incomplete forms of thecomplex (Govezensky and Zamir, 1989). B. Extracts electrophoresed underdenaturing conditions and immunoblotted with anti GroEL antibodies.Lane 1, E.coli T850; lane 2, E.coli T850 transformed with pDGlO; lane 3,E. coli KL355; Lane 4, E. coli T850 transformed with pKT200.

*i 5;!;:K .s 2 ' 'i..4 iI

Fig. 2. Accumulation of native MoFe protein and its structural polypeptidesNifDK in Kpneumoniae transformed with pDG10. Nontransformed (-)and pDG1O transformed (+) wild type Kpneumoniae cells were nif-derepressed for the periods indicated and analysed for native MoFe protein(upper panel) or denatured NifDK polypeptides (lower panel) as describedin the legend to Figure 1 and Materials and methods.

gene(s) sugE, for suppressor of groEL, was mapped to aDNA fragment containing two nested in-frame ORFs. Atranscript starting upstream of both ORFs was predominantin cells growing logarithmically at normal temperatures.Another transcript, induced in heat-treated or stationaryphase cells independently of a32, was initiated within thelarger ORF upstream of the smaller ORF. The results suggestthat sugE encodes a novel chaperonin-related function,potentially modulated by growth conditions.

Results

Demonstration of a groEL suppressor gene inpLC1643In order to examine the potential role ofgroEL in biologicalnitrogen fixation, we have previously compared wild type

890

Fig. 3. Effect of pDGIO on bacteriophage T4-induced cell lysis. The non-transformed E. coli (Ec) strains and transformants with the specified plasmidswere analysed at the temperatures indicated for bacteriophage T4-inducedcell lysis by the spot test described in Materials and methods.

(strain KL355) and a groEL mutant (strain T850) of E. coliboth transformed with pWK220, a plasmid containing theentire nifgene cluster of K.pneumoniae (Govezensky et al.,1991). Following derepression of the nif regulon,accumulation of the MoFe protein was assayed byfractionation of bacterial crude extracts on non-denaturinganaerobic gels followed by immunoblot analysis. The resultsindicated that the groEL mutation brought about a drasticlowering in the accumulation of both MoFe protein and itsconstituent polypeptides without altering the non-denaturingelectrophoretic migration that reflects the state of MoFeprotein assembly. Co-transformation of the groEL mutantwith a plasmid containing the E. coli wild type groE operonreversed the mutant phenotype.A similar assay was used to examine the possible presence

of a groEL suppressor gene in pLC 16-43 (Figure IA). Asobserved previously, transformants of the groEL mutant withthe nif gene cluster accumulated very little, if any, MoFeprotein. However, when co-transformed with pDG1O, aplasmid containing a 2.3 kb HindmI-EcoRI subfragmentof the insert in pLC16-43, the cells accumulated wild typelevels of the nitrogenase component. The reversal of themutant phenotype in the co-transformants was not associatedwith an increase in the GroEL level (Figure 1B).Another experiment, also modeled after our earlier study,

was based on the finding that the normal levels of GroEproteins in K.pneumoniae limit the rate of nif derepression(Govezensky et al., 1991). Accordingly, wild typeK.pneumoniae cells were compared with transformants withpDG10 for the kinetics of accumulation of native MoFeprotein and its constituent polypeptides after nifderepression.The results (Figure 2) indicated that the accumulation of boththe native forms of MoFe protein and the constituentpolypeptides started earlier and progressed faster in thetransformed as compared with the non-transformed cells.A similar response has been observed earlier withK.pneumoniae transformed with the E.coli groE operon.The groEL mutation in E. coli T850 was originally shown

to abolish T-even phage propagation due to impaired phagehead formation (Takano and Kakefuda, 1972). The abilityof sugE to suppress the phage morphogenetic defect in thegroEL mutant was examined in assays of bacteriophageT4-induced cell lysis. A qualitative analysis (Figure 3) firstshowed that transformation with pDG1O, similar totransformation with pKT200, a plasmid carrying the wildtype groE operon, restored the ability of the host cells toaccommodate phage growth. The two types of transformantsdiffered, however, in their temperature response: whiletransformation with pKT200 permitted complete lysis at30°C, the function provided by pDGIO was not expressed

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A novel multicopy suppressor of a groEL mutation

Table I. Effect of pDG1O on the plating efficiency of bacteriophage T4

Bacterial host Plating efficiency at

300C 370C 42°C

KL355 1 1 1T850 < 10-7 < 10-7 < 10-7T850 + pDG10 <10-7 l0-4 10-2

Efficiency of plating is expressed as the lowest concentration of phageyielding visible plaques on the hosts T850 and T850 + pDG1O relativeto the corresponding concentration for the wild type strain KL355.

93 9 4 95 [min]

/\~sugEampCsfrdgf!~- -.*L *- 4I I if

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E

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sugE

ORF1

H

amp

I I ~I 1~ I_ IE _ =: ORF2 -

o (a _ E X 0 'a_0.0 co L

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iiii i~~~~~~~~~~~~~~~~~~~

0 bp

Fig. 4. Genetic and physical map of the sugE chromosomal locus. Thelocation of sugE is shown on the genetic map of Jenkins et al. (1986). Thelocation of restriction sites within the 2.0 kb PstI-HindlII fragment havebeen verified by nucleotide sequence determination.

at this temperature, but only at higher temperatures. Lysiswas evident at 37°C and more pronounced at 40°C, a

difference not clearly visible in the photograph shown. Amore quantitative estimate of sugE function was obtainedby a comparison of the phage plating efficiency at thedifferent temperatures (Table I). With T850 cellstransformed with pDGIO as hosts, raising the temperaturefrom 37°C to 42°C brought about a 100-fold increase inplating efficiency. At 42°C, plating efficiency on thetransformants was in the order of 1 % of the plating efficiencyon the wild type KL355 strain.The ability of sugE to suppress groEL mutations other than

that in strain T850 was examined by assaying cell lysis bybacteriophage T4 or bacteriophage X. With the groEL-strains CG2241 (groEL44) and CG2243 (groEL140) (Tillyet al., 1981), transformation with pDG1O enabled cell lysisby both types of bacteriophage. These results indicate thatsuppression by sugE is not limited to a single groELmutation.

The ability of sugE to suppress groES mutations was alsoexamined. Transformation with pDG1O of the groES-strains CG2244 (groES619) and CG1921 (groES30) (Tillyet al., 1981) did not enable cell lysis by bacteriophage X,that unlike T4 is groES-dependent (Zeilstra-Ryalls et al.,1991). Hence, sugE is unable to correct the defect in phageassembly due to these groES mutations.

Nucleotide sequence and predicted ORF(s) of sugEThe nif and phage-induced lysis assays provided strongsupport for the presence of a multicopy suppressor ofgroEL(sugE, for suppressor of groEL) in the HindIH-EcoRIfragment of pLC16-43 subcloned in pDG10.To localize more precisely the span of sugE within the

2.4 kb insert in pDG10, subclones that had been generatedby using several restriction enzymes (Figure 4) wereexamined for suppressor activity in bacteriophageT4-induced T850 cell lysis. The 1.4 kb PstI fragment, whichcontains the 3' end of ampC and the 1.4 kb SmaI-EcoRIfragment, which is free of ampC (Jaurin and Grundstrom,1981), both showed suppressor activity. Therefore, sugEresides within the sequence shared by the two fragments,i.e. the 1.0 kb SmaI-PstI fragment. The complete nucleotidesequence was determined for the 1.4 kb PstI fragment andtwo nested in-frame ORFs were formally identified withinthe sequence flanked by the SmaI and PstI restriction sitesthat includes sugE (Figure 4).The nucleotide sequence including the two ORFs and

flanking sequences is shown in Figure 5A. ORFI encodesa 178 amino acid polypeptide of 18 922 Da. A potentialShine-Dalgarno sequence starts at position -13 withrespect to the initiation codon (ATG1). The potentialpresence of another, in-frame nested ORF was first suggestedby a database search, which indicated the presence in theProteus vulgaris chromosome of an ORF encoding apolypeptide closely similar to the predicted sequence ofORF1 extending from Met74 to near the carboxy-terminus(Figure 5B) (Cole, 1987). An examination of the sequenceupstream of ORF2 revealed a potential Shine-Dalgarnosequence starting at nucleotide position -7 from the potentialinitiation codon (ATG2). ORF2 encodes a 105 amino acidpolypeptide of 10 900 Da. No similarity was found betweenthe sequence upstream of ORF2 (including the sequenceencoding the N-terminal extension of ORFl and the5 '-untranslated region) in E. coli and the sequence upstreamof the ORF2 homolog in P.vulgaris (Cole, 1987).

Southern blot hybridization verified the presence of sugEin the E. coli chromosome and demonstrated the conservationof at least part of the gene in Kpneumoniae (data not shown).

Translational products of sugEDirect support for the presence of two ORFs in sugE wasprovided by observing the corresponding translationalproducts in transformants with pTG31, a plasmid derivedfrom a recombinant containing the PstI fragment includingsugE (Figure 4) cloned dov'nstream of the T7 promoter ina Bluescript vector. In pTGS 1, exolH/mung bean nucleasedigestion was used to delete the sequence upstream of sugEto 53 bp upstream of ORF1. Following the induction of thechromosomally located T7 RNA polymerase gene andsubsequent addition of rifampicin to inhibit the E. coli RNApolymerase (Studier and Moffatt, 1986), two polypeptidesof 19 and 11 kDa were preferentially labelled with

891

No


APi

-3; -I1C lvw VVY1 AAAATCATCAGATTCCCATCATTTTTGGCGATGTTGTCTATTATTAATTTGCTATAGGCAAACATAAATAA

72 CATTACCTAAAAGGAAGACGTTATGGTGAAGAAGACAATTGCAGCGATCTTTTCTGTTCTGGTGCTTCAAC

SD

PvuII4

ATG GCGGTAACGCGATTTM A V T R F

L A Q Q R K R S N K Q R Y D S C V V P F V F S AP2

282 GATAGTCACAAAGGTAATAGTTGAAATTCCCCTGCCACCTGGCAAAATATCCGTTCAACCATCAGCTTTGC 54I V T K V I V E I P L P P G K I S V Q P S A L Q

MroI SD353 AGGACGACCTGCAAACGCCTCTTTTCACCGGGGACGGCCCCAATTCTCCGGAGCCTGAT ATG TCCTGGA 4 77

D D L Q T P L F T G D G P N S P E P D M S W I

422 TTATCTTAGTTATTGCTGGTCTGCTGGAAGTGGTATGGGCGGTTGGCCTGAAATATACCCACGGCTTTAGT 27 100I L V I A G L L E V V W A V G L K Y T H G F S

493 CGTTTGACGCCGAGTGTTATTACTGTGACGGCGATGATTGTCAGTATGGCGCTACTTGCCTGGGCGATGAA 51 124R L T P S V I T V T A M I V S M A L L A W A M K

564 ATCGTTACCAGTAGGGACGGCTTATGCCGTGTGGACGGGTATTGGCGCCGTCGGCGCGGCCATAACCGGCA 75 148S L P V G T A Y A V W T G I G A V G A A I T G I

635 TTGTGCTGCTCGGTGAGTCCGCTAACCCGATGCGCCTGGCGAGTCTGGCGTTAATCGTATTGGGGATTATT 98 171V L L G E S A N P M R L A S L A L I V L G I I

706 GGTCTGAAACTCAGCACTCAC TAA CTACCAGGCTGCTGTACCCAAATAAATTTACTGACATCAAACCCT 105 178G L K L S T H STOP

SmaI774 TCCCGGGTCGCGA

BEc SugE MAVTRFLAQQ RKRSNKQRYD SCVVPFVFSA IVTKVIVEIP

vRFEc SugE LPPGKISVQP SALQDDLQTP LETGDGPNSP EPDMSWIIL-

l l llPv OrfD MSWI ILF

Ec SugE VIAGLLEVVW AVGLKYTHGF SRLTPSVITV TAMIVSMALL1 11111:11 1111111111 :11111:11: :111111: 1

Pv OrfD V-AGLLEIVW AVGLKYTHGF TRLTPSIITI SAMIVSMGML

Ec SugE AWAMKSLPVG TAYAVWTGIG AVAAAITGIV LLGESANPMR11111 1111:11111 11: 11 11: 11111

Pv OrfD SYAMKGLPAG TAYAIWTGIG AVGTAIFGII VFGESANIYR

Ec SugE LASLALIVLG IIGLKL-STH*

lI III 111111 1Pv OrfD LLSLAMIVFG IIGLKLAS*

Fig. 5. Nucleotide and deduced protein sequence of sugE (EMBL data library accession number X69949). A. The nucleotide sequence and deduced aminoacid sequence of ORFI and of ORF2 (residue numbers on the right) are indicated below the corresponding codons. Putative Shine-Dalgarno sequencescorresponding to ORFI and ORF2 are underlined and designated as SD. Start sites of transcription from the P1 and P2 promoters are indicated by wavyarrows. Putative -10 and -35 elements of u70-dependent promoters in P1 are underlined. B. Alignment of the sugE ORF (Ec SugE) with the OrfD productfrom P.vulgaris (Pv OrfD) (Cole, 1987). The starts of ORFI and ORF2 are indicated. Vertical bars, amino acid identities; colons, highly conservativereplacements.

[35S]methionine (Figure 6). These polypeptides, closelysimilar in size to the predicted products of ORFI and ORF2,were not observed when the insert was cloned in the oppositeorientation to the T7 promoter (data not shown). BecauseORF1 and ORF2 encode for nearly the same number ofmethionine residues, [35S]methionine labelling of the twopolypeptides directly reflects their relative rates of synthesis.The results therefore indicate that when expressed from theT7 promoter the predominant translational product of sugE,as cloned in pTG31, is ORF1.

1 2

..._ ..

...........

........ _0.; .Xt ..:..:X

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=r

Transcriptional regulation of sugEThe E. coli strains studied were found to contain considerablelevels of sugE transcripts permitting us to use non-transformed cells for nuclease S 1 analyses. The possibility,suggested by the assays of T4-induced lysis that sugE washeat-regulated, warranted the analysis of cells exposed todifferent temperatures. Initially, RNA was isolated fromKL355 cells growing logarithmically at 30°C, or after theirexposure to 45°C for 30-45 min. (A subsequent analysisof sugE transcripts in the groEL mutant T850 revealedsimilar transcriptional activity, start sites and temperaturedependence as observed in the KL355 strain.)The results (Figure 7A) indicated the existence of different

preferential sites of transcription initiation at 30°C and 45'C.The major transcript at 30°C was initiated at position -145with respect to ATG1 (Figure 7B). The promoter dictatingthis transcription, P1 (Figure 7D), includes sequence motifscorresponding to consensus -10 and -35 elements of(0-dependent promoters (Figure 5) (Cowing et al., 1985).Within 15 min after the cells have been transferred to 45°C(Figure 7A and C), sugE transcription from P1 was reducedto a non-detectable level and another transcript initiatedwithin ORF1, 82 bp upstream of ORF2, which is barelyevident at 30°C, was very slightly increased in amount. Theintensity of this transcript rises most markedly after 45 min892

1l9kD

L~~~~I k

Fig. 6. In vivo detection of the sugE translational products. E coli BL2I/DE3transformed with pTG31 (described in the Results) was grown at 30°C inM9 medium to A595 of 0.5. Expression of T7 RNA polymerase wasinduced by the addition of 0.5 mM IP`TG. After 30 min incubation, rifampicinwas added to 200 ,ug/ml and following 30 min incubation, [35S]methioninewas added to 50 yCi/ml and incubation was continued for 10 min. Proteinextracts were resolved on SDS-PAGE and the gel was autographed. Lane 1,extract of cells not treated with IPTG and rifampicin; lane 2, extract ofcells treated with IPTG and rifampicin. The estimated sizes of the sugEtranslational products are indicated next to the arrows marking their position.

at 45°C. The conclusion that the transcript observed in theheat-shocked cells arises from an independent promoter, P2,is directly supported by the analysis of cells subjected to heat-


(A) B 2 32

{A) 2~ o. 4 i,;

P -

-~~~~~~~~~~~~~~~ 2P2~~~~F-.--.

(D) P2 _ ...

_* -~

Fig. 7. Nuclease SI analysis of sugE transcription start sites. Isolation ofRNA and analytical procedures were as described in Materials and methods.A. Nuclease SI analysis using as probe fragment II shown in D, with RNAfrom E.coli KL355 growing logarithmically at 30°C (lane 1), after 45 minexposure to 45°C (lane 2), after 15 min exposure to 45°C (ane 3), or grown

at 30°C to stationary phase (lane 4). B. Precise localization of the sugEtranscripton start site in the RNA analysed in lane 1 of (A). Lane 1, nucleaseSI analysis using as probe fragment I shown in D; lane 2, A + G productsof a sequencing reaction of the probe (Sambrook et al., 1989). C. Preciselocalization of the sugE transcription start site in the RNA analysed in lane 2of A. Lane 1, nuclease SI analysis using as probe fragment II shown inD; lane 2, A+G products of a sequencing reaction of the probe. D. Theprobes used in the analyses in relation to coding and regulatory regionsin sugE. P1, P2 in A-C, fragments protected by transcripts from the P1and P2 promoters, respectively.

shock in the presence of rifampicin (data not shown). Nosequence motifs clearly related to consensus elementsrecognized by u7O or the heat-shock-mediating sigma factorsU32 (Cowing et al., 1985) and j24 (Erickson and Gross,1989; Wang and Kaguni, 1989), were discerned within thesequence corresponding to the P2 promoter (Figure 7D).

Transcription of sugE was also analysed in stationary phasecells growing at 30°C. The results (Figure 7A) revealedconsiderable transcriptional activity from the P2 promoterin these cells, in contrast to the negligible activity inlogarithmically growing cells. The activation of the P2promoter in the stationary phase cells was not accompaniedby a drastic decrease in transcriptional activity from theupstream promoter.Although P2 lacked motifs characteristic of u32-dependent

promoters, the possible involvement of a32 in transcriptionfrom this promoter was examined by analysis of sugEtranscripts in R40NL8, a ArpoH strain. The strain studiedcontained the suhX suppressor (overexpression of the groEoperon) (Kusukawa and Yura, 1988), but was still unableto grow at the elevated temperatures normally required foractivation of the P2 promoter. In view of this difficulty, we

Fig. 8. Nuclease SI analysis of sugE transcription start sites in a A rpoHmutant and isogenic wild type strain. The RNA analysed was isolated fromlogarithmically growing strains RNL8 (ArpoH) (lane 1) and MC4100 (wildtype) (lane 2), or from stationary phase cultures of RNL8 (lane 3) andMC4100 (lane 4). P1 and P2, fragments protected by transcripts from theP1 and P2 promoters, respectively.

attempted to examine transcriptional activity from P2 notin heat-treated, but in stationary phase cells. The analysis(Figure 8) indicated that in stationary phase, transcriptionfrom P2 in the ArpoH strain did not differ from that in thecorresponding wild type strain MC4100. In both strains, P2transcripts constituted the major transcriptional products ofsugE.The ArpoH mutant and wild type isogenic strain were also

analysed during logarithmic growth at 30°C (Figure 8).The two strains contained higher levels of P1 transcripts andrelatively lower levels of P2 transcripts compared withstationary phase cells. The two strains differed, however,when compared for the ratio between the two transcripts.In the wild type cells, P1 transcripts were in excess to P2transcripts (as shown above for logarithmically growingKL355 cells), whereas in the mutant, P2 transcripts weremore abundant than P1 transcripts.

DiscussionThe multicopy groEL suppressor function of sugE was firstobserved in an assay monitoring the synthesis and assemblyof the MoFe protein component of K.pneumoniaenitrogenase. Plasmid-borne sugE restored to a nif-transformed E. coli groEL mutant the ability to accumulatehigh levels of the nitrogenase component. When transformedinto wild type K.pneumoniae, sugE accelerated the processof MoFe protein biogenesis after derepression of the nifregulon. The functions manifested by sugE in the two nifassays were indistinguishable from those previously observedin transformants with the wild type groE operon. Hence,sugE not only suppressed a groEL mutation in E. coli, butalso mimicked the effect of groE overexpression in aK.pneumoniae wild type background.

Bacteriophage T4 is unique among phages requiring groEL893

DRz--1 t-

1-11,OR..-2

m zz> Z.a- <


for their morphogenesis in its independence of groES(Zeilstra-Ryalls et al., 1991). Bacteriophage T4-induced celllysis can then serve as a measure for GroEL functionindependent of its interaction with GroES, althoughdependent instead on the T4 gene 31 product (Zeilstra-Ryallset al., 1991). Plasmid-borne sugE also enabled lysis ofgroEL mutants by bacteriophage X that requires groES(Zeilstra-Ryalls et al., 1991). The ability of sugE to restorephage growth in groEL mutants proves that the scope of sugEfunction transcends the nif system and includes phagemorphogenesis, the 'classical' groE function.The possibility that the single continuous ORF in sugE

encodes for two polypeptides was first raised by theconservation in P. vulgaris of ORF2, but not the N-terminalextension specific for ORF1. The ORF2 homologue inP. vulgaris, OrfD, forms one of four ORFs founddownstream and in opposite orientation to the frd operon,as shown in a sequencing analysis of this chromosomalregion (Cole, 1987). No evidence has been obtained for thepresence in P. vulgaris of an ampC gene (Cole, 1987), whichin E. coli separates the frd operon from sugE (Bachmann,1990). This, as well as other variations indicate aconsiderable divergence in this region in differententerobacteria. Because the E. coli sequence upstream of thesugE ORF2 shows no similarity to the P. vulgaris OrfA, OrfBor OrfC, we conclude that the genetic elements conservedin this chromosomal locus between the two enterobacteriaare the frd operon and the oppositely oriented OrfD/ORF2of sugE.The presence of two functional ORFs within sugE was

directly demonstrated by the in vivo synthesis of twopolypeptides from sugE transcribed in its entirety from theT7 promoter. Under these conditions, the polypeptideencoded by ORFI is synthesized in excess to the shorter,ORF2-encoded polypeptide. A 60 min chase with non-labelled methionine did not alter the ratio between the 19and 11 kDa polypeptides (data not shown), ruling out thepossibility that the shorter polypeptide was a breakdownproduct of the longer polypeptide.The selective expression and regulation of each of the

potential translational products of sugE is made possible bythe use of two, differentially regulated promoters. Analternative possibility that the P2 transcript arises by post-transcriptional processing of the P1 transcript, is notsupported by the course of change in transcript levels aftertransfer of the cells to 45°C. After 15 min at this temperaturethe P1 transcript disappears, whereas the P2 transcript isbarely detectable and is fully induced only later.

Expression from the P1 promoter upstream of ORF 1,characteristic of cells growing at 30°C, would yieldpredominantly the larger polypeptide. At more elevatedtemperatures or in stationary phase cells, the activation oftranscription from the P2 promoter and the correspondingdecline in transcription from the P1 promoter, would resultin an increased synthesis of the shorter polypeptide.

Transcription from the P1 promoter probably occursconstitutively in cells growing logarithmically at normalphysiological temperatures. Based on the nucleotide sequenceupstream of the transcription start sites, P1 includes elementstypical of u70-dependent promoters. Transcription from theP2 promoter is induced by high temperature or in stationaryphase cells. These two modes of induction could employ thesame or different types of sigma factors.

In a previous study (Jenkins et al., 1991), the synthesisof several heat-inducible proteins in E. coli, i.e. DnaK,GroEL and HtpG, has been shown to be stimulated in cellsstarved for carbon. Induction by starvation resembled heat-induction in its dependence on a32. By analogy, a singlesigma factor could be involved in both heat and stationaryphase induction of sugE. The observation made in the presentstudy that stationary phase induction of transcription fromP2 is not altered in a A rpoH strain eliminates its regulationby a32 under these conditions. Nevertheless, the possibilitythat thermal activation of P2 still requires a32 cannot becompletely dismissed. While we have not determined yetthe nature of the sigma factor(s) involved in transcriptionfrom P2, this promoter might belong to a hithertounrecognized heat-inducible regulon.An intriguing feature of sugE transcription in the ArpoH

strain is the relatively high level of P2 transcripts in cellsgrowing at 30°C. Possibly, in the absence of a32 anddespite the overexpression of groE (due to suhX), atemperature of 30°C is perceived by the cells as stressful,thus leading to the generation of a signal(s) for P2 activation.By affecting the relative transcriptional activity from the

P1 and P2 promoters, temperature and growth phase wouldbe expected to alter the ratio between the ORFI and ORF2products. The activity of sugE in the nif system wasdemonstrated at 30°C, an assay temperature dictated by thethermosensitivity of NifA, the transcriptional activator of theK.pneumoniae nif regulon. In contrast, the block in phagegrowth was not significantly overcome by sugE unless thetemperature was raised above 37°C. The plating efficiencyobserved at 42°C may not be maximal and might furtherincrease by rising the temperature to 45°C where sugEtranscription from P2 is most effective. The two systemsalso differed in that nifderepression was routinely assayedin stationary phase cells, whereas bacteriophage growthoccurred in growing cells. Stationary phase, similar to hightemperature, would induce transcription from P2 andconsequently favour translation of ORF2. However, residualtranscriptional activity from P1 is likely to provide someORF1 product even under these conditions.The functional significance of the two ORFs in sugE

remains unclear. By its size and amino acid sequence, theN-terminal extension encoded in ORFl is not likely to serveas a proteolytically-removed signal peptide for secretion.Furthermore, pulse -chase analysis did not reveal any time-dependent breakdown of the 19 kDa into the 11 kDapolypeptide (data not shown). The ORF1 and ORF2polypeptides might function independently of each other,form a complex or act in succession. In all instances, thechanging ratio between the two proteins as a function ofphysiological condition would be expected to modulate theiractivity.The conservation of at least part of sugE in several

enterobacteria (E. coli, P. vulgaris and K.pneumoniae)together with the considerable transcriptional activityobserved in non-transformed E. coli suggest that this genefulfils an important cellular function. Among severalpossibilities, sugE function might be that of a chaperoninsystem normally showing affinity for a subset of cellularproteins poorly recognized by GroEL. Overexpression maythen compensate for the lower affinity and allow the sugEproduct(s) to functionally replace GroEL. Another possibilityis that sugE product(s) interact with GroEL to facilitate or

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modify its action. Possible functional modulation of GroELby ApppA binding or phosphorylation have been recentlyproposed (Johnstone and Farr, 1991; Sherman and Goldberg,1992). It should, however, be stressed that the groELsuppressor and mimetic activities of sugE were not foundto affect the level and electrophoretic mobility of native ordenatured GroEL. The fact that transformation with pDG10did not enable lysis ofgroES mutant strains by bacteriophageX suggests that sugE does not fully circumvent the groEsystem but may still require groES function.The two proposed models of sugE activity are in line with

the mechanisms of sugE regulation permitting bothconstitutive and stress-induced expression, dependent on astill undefined sigma factor. This regulatory pattern isgenerally similar to that of the groE operon that is expressedalternatively from a u70-dependent constitutive promoter ora stress-responsive, c32-dependent promoter (Zhou et al.,1988). The intriguing feature of sugE is that transcriptionfrom the two promoters not only serves as a transcriptionalregulatory device, but potentially yields different translationalproducts. Further studies of the regulatory mechanisms ofsugE and the function(s) filfilled by its product(s) would shedlight on a potentially novel molecular chaperone.

Materials and methodsBacterial strains and plasmidsThe bacterial strains used in this study include Kpnewnoniae wild type andthe following E.coli strains: KL355 [CGSC strain 4296; tonA2proA44 lacYlsupE44(?) galK2 X- hisGI rflDI galP63 malTI xyl-7mtl-2 argHl ampCplpurA44) (Takano and Kakefuda, 1972); T850 (CGSC strain 4812; tonA2proA44 lacYl supE44(?) gal-6 X- hisGI rpsL9 malTI xyl-7 mtl-2 thi-IargHl mop-i (synonym of groEL-)] (Takano and Kakefuda, 1972);MC4100 [F- araDJ39 A(argF lac) U169 rpsLISO relAl flbBS301 deolptsF25 rbsR]; R40NL8 [F- araDJ39 A(argF lac) U169 rpsLJSO relAlflbBS301 deol ptsF25 rbsR ArpoH30::kan zhfSO::TnlO suhX401(overexpression of groE)], a derivative of KY1603 (R40-1) (Kusukawa andYura, 1988) kindly provided by Z.Livneh; BL21/DE3 (a lysogen of BL21containing a chromosomal gene for T7 RNA polymerase under the controlof the lacUV5 promoter) (Studier and Moffatt, 1986). Plasmids used were:pLC16-43 (assigned genes: mop ampCfrdAfrdB) (Neidhardt et al., 1983);pKT200 (containing the wild type groE operon from E.coli) (Bloom et al.,1986); pWK220 (containing the Kpneumoniae nifgene cluster) (Puhler andKlipp, 1981); pDGIO (containing a 2.3 kb HindIl-EcoRI subfragmentof pLC16-43 cloned in pUC18).

Growth conditions and nitrogenase derepressionKpneumoniae was normally grown in NFDM medium containing 0.2%ammonium acetate at 30°C. When assayed for nif, transformants of E.colistrains KL355 and T850 were grown similarly to Kpneumoniae, but themedium was supplemented with 25 itg/ml each of L-proline, L-arginine,L-histidine, adenine and thiamine. In other instances these two strains, aswell as the other E. coli strains used in this study were grown in LB at 300C.Depending on the transforming plasmids, the medium contained: ampicillinat 400 itg/ml for Kpnewnoniae and at 100 jg/ml for E.coli; chloramphenicolat 15 Ag/ml (Govezensky and Zamir, 1989; Govezensky et al., 1991).Conditions for nitrogenase derepression of stationary phase cultures ofK.pneumoniae and nif-transformed E.coli were essentially as describedpreviously by Berman et al. (1985a) and Govezensky et al. (1991).

Anaerobic protein extractionThe procedure used for the extracton of Kpneumoniae and nif-transformedE. coli for the analyses of native MoFe protein was essentially as previouslydescribed by Govezensky and Zamir (1989) and Govezensky et al. (1991).

Gel electrophoresis and immunoblottingThe procedures for denaturing and non-denaturing gel electrophoresis andimmunoblotting (using [ 125I]protein A) were essentially as described(Govezensky et al., 1991; Govezensky and Zamir, 1989). Blots were probedwith rabbit polyclonal antibodies raised against MoFe protein fromKpneumoniae or against GroEL from E.coli.

Cell lysis by bacteriophage T4Transformed and non-transformed E.coli cells were assayed for lysis bybacteriophage T4 by a spot test or by determining the lowest serial dilutionyielding visible plaques essentially as described (Revel et al., 1980). In thespot assay, before photography the plates were treated briefly with Coomassieblue and immediately washed with water. Under these conditions, the dyeadsorbed to all areas of the plate except to whole cells.

Nucleotide sequence determinationDNA sequence analysis was performed by the chain termination methodemploying T7 DNA polymerase (Sequenase, USB).

RNA isolation and nuclease Si analysisIsolation of total bacterial RNA and nuclease SI analyses were performedas described (Ausubel et al., 1987; Sambrook et al., 1989). Nuclease SIanalyses were performed with 50 jg of RNA.

AcknowledgementsWe are grateful to M.Berlyn, C.Georgopoulos and Z.Livneh for bacterialstrains and plasmids. This research was supported by grant No 90-00134from the United States-Israel Binational Science Foundation (BSF) Jemsalem,Israel and by The Leo and Julia Forchheimer Center for Molecular Genetics,Weizmann Institute of Science.

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Received on August 4, 1992; revised on December 9, 1992

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a novel multicopy suppressor of a groel mutation from different

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