MolecularMrcrobialogy (1991)5(5), 1285-1293 ADONIS 0950382X9100142P

The hha gene modulates haemolysin expression inEscherichia coli

J. M. Nieto,^ M. Carmona.^ S. Bolland,^ Y. Jubete,^F. de la Cruz^ and A. Juarez'*'Departamento de Microbiologia, Facultad de Biotogia.Universidad de Barcelona, Avenida Diagonal 645, 08071Barcelona, Spain.^Departamento de Biologia Molecular, Facultad deMedicina. Universidad de Cantabria, Cardenal HerreraOria. s/n. 39011 Santander. Spain.

Summary

A mutation in the hha allele results in a large increasein the production of intraceliular as well as extra-cellular haemotysin in Escherichia coli cells harbour-ing the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between490 and 491.6 kb on the physical map of the E. colichromosome. From the DNA sequence of hha a smallpotypeptide of 8629 Da was predicted and wasexpressed in minicells. The deduced polypeptidesequence did not show significant similarities to othercharacterized proteins related to the regulation ofgene expression in E coli, although it was shown thatthe hha mutation increases cytoplasmic synthesis ofhaemotysin.

Introduction

The Escherichia coli haemolysin is frequently associatedwith strains isolated from extraintestinai infections. Thistoxin belongs to a family of related proteins that aresecreted by a mechanism different from the A/-terminalsignai-sequence-dependent pathway. This familyincludes the related cytotoxins produced by Pasteurellahaemolytica (Strathdee and Lo. 1987) and Bordetellapertussis (Glaser et ai, 1988) and they share someconserved features in the secretory mechanism, as well asa common genetic organization. Although interesting as avirulence factor (Welch ef al.. 1981). this cytotoxin is alsostudied because of the unusual mechanism by which it isexported (Juarez and Goebel. 1984; Koronakis et al..1989; Mackman et al.. 1986. 1987). being one of the fewexamples of a protein secreted to the external medium byE. CO//(Hollander a/., 1986).

Received 2 November. 1990; revised 23 January, 1991. "ForconBSpond-ence. Tel. (343) 4110592; Fax (343) 4110592.

A transcriptional unit. hlyCABD. codes for the synthesisand secretion of haemolysin. A major 4 kb hlyCA and aminor 8kb hlyCABD transcript appear to be the maintranscripts. Both start at the same promoter, located some400bp upstream of hlyC. and the minor 8kb transcript isthought to be produced by readthrough, or antitermi-nation. at a rho-independent transcriptional terminatorlocated between hlyA and htyB (Welch and Pellet. 1988;Koronakis et al.. 1988). The hlyA gene codes for apolypeptide which, when activated by the hlyC geneproduct, becomes the active haemolysin (Nicaud et al.,1985). Secretion of haemolysin is somehow directed bythe products of genes hlyB and hlyD (Wagner ef al., 1983;Mackman et al.. 1985).

Recently, the sequence hlyR was characterized duringanalysis of the haemolysin determinant harboured by thehaemolytic plasmid pHly152. This c/s-acting 600 bpsequence is located 1500bp upstream of hlyC, and isessential for the production and secretion of haemolysincoded by plasmid pHly152 (Vogel etal., 1988). Cloning ofthe hlyCABD cluster into pACYCi84 gave the recombi-nant plasmid pANN202-312. which produced haemolysinpoorly, especially in the external medium (Goebel andHedgpeth, 1982; Juarez ef al., 1984). When this fragmentincluded the hlyR sequence, the new recombinant plas-mid expressed large amounts of intracellular as welt asextracellular haemolysin (Gonzalez-Carrero ef al.. 1985;Vogel et al.. 1988). When we examined the poor haemo-lysin expression in cells harbouring pANN202-312, weisolated a mutation in the E. coli chromosome whichresulted in high expression of both intracellular andextracellular haemolysin (Godessart et al., 1988). Appar-ently this mutation, termed hha, could compensate forhlyR and was mapped at min 10.5 on the £ coli chromo-some. Interestingly, the hha mutation also affected theexpression of other heterologous proteins cloned in E coli(Nieto ef al.. 1987). In the present report we furtherinvestigate the rote of the hha mutation by cloning andsequencing the hha gene, identifying the gene product,and analysing its effect on the synthesis of haemolysin.

Results

Cloning strategy and sucbloning of the hha gene

Strain E. co/; Hha-2 contains a Mu d l insertion in the hhagene (Godessart et ai. 1988). We cloned the hha gene by

1286 J. M. Nietoe\a\.

BB PVGVVE ETT T TT TTf f

Plasmid

PUBM5005-I

PUBM5OO5-2

pUBM5005-3

PUBM5005-4

PUBM5005-5

PUBM20

PUBM2I

pUBM22

PU8M23

BBTTBBTTBBTT

ETET

ET

ETETET

I kb

Fig. 1. Cloning and subclonrng of the hha gene. Filled lines show theextent and restriction sites of the insert D̂ 4A of representative Hha'clones (clones pUBM5005-1 to -5) in a mini-Mu 5005 gene libraryobtained from strain C600 (the vector replicon Mini-Mu d5005 is notshown), and the DNA fragments used >or subcloning the hha gene,rendenng. respectively, plasmids pUBM20 to pUBM23. B. Bs/nHI; E,EcoRI. G. BglW, P. Psll. V, EcoRV.

preparing a tysate of the Hha^ strain E coli C600 Mu cts(pEG5005) and used it to infect £ coti Hha-2(pANN202-312). The transductants grown on biood-agar platessupplemented with ampicillin. chloramphenicol. andkanamycin were screened for the ability to complementthe t)ha mutation, that is, to drastically reduce the largehaemolysis halo produced by E. coli Hha-2(pANN202-312) colonies and five different complementing cloneswere selected. Their external haemolytic activity was0.004 units, whereas the external haemolytic activity of theHha-2 strain was 15.3 units. Upon curing the haemolysis-conferring plasmid. pANN202-312, plasmid DNAs(PUBM5005-1 to PUBM5005-5) were isolated and charac-terized by EccR\- and SamHI mapping {Fig. 1). Althoughcontaining different sizes of inserts as well as in thechromosomal sequence, these plasmids contained anoverlapping region of about 6kb which, in the originalstrain, was flanked by EcoRI and SamHi sites. Thecommon fragment, obtained from pUBM5005-2, wasinserted into pBR325. yielding plasmid pUBM20 (Fig. 1),Transformation of E. coli Hha-3(pANN202-312) with thisplasmid confirmed that the complementing activity wascontained in the subcloned fragment. Further restrictionanalysis of pUBM20 allowed us to map two Pst\ restrictionsites in the EcoRl-SamHI fragment. Plasmid pUBM21was obtained by inserting a 3.2kb EcoRI-PsM fragmentinto pBR325 (Fig. 1). Again, EcoRV mapping allowedfurther subcloning by inserting an 1.7 kb EcoP,y~EcoP,yfragment in pBR322. rendering the plasmid pUBM22.which was still capable of complementing the hhamutation. Insertion of the omega cassette (Prentki and

Khsch, 1984) in a BgtW site vi/ithin the fragment cloned inpUBM22 did not destroy the complementing activity. Thuswe obtained plasmid pUBM23, containing the 1.4 kbSg/ll-EcoRV fragment cloned in pBR322 (Fig. 1).

Additional evidence indicating that the cloned fragmentcontaining the hha sequence was obtained upon cloningan EcoRI fragment from E coli Hha-3 ihha::Tr\5phoA)including the kanamycin resistance (Km^ determinantfrom the transposon into pBR325. Restriction analysis ofthe resulting Km" clones (not shown) indicated that E colistrain Hha-3 contains TnSphoA inserted in the Sg/ll/EcoRV fragment common to plasmids pUBf\fl20,PUBM21, pUBM22, and pUBfyl23.

Localization of the hha gene on the physical map of theE. CO//chromosome

Since Kohara etal. (1987) published the physical map ofthe whole E. co//chromosome, genetic and physical mapscan be correlated. We tried to localize the 5.6kb EcoRI-SamHI fragment containing hha on the E coti physicalmap. A coinciding restriction sequence was tound in the10.4 min area of the E coli restriction map. confirming ourgenetic mapping data (Godessart et al.. 1988). Thus, thehha gene must be located on the EcoR'^-EcoVW fragmentbetween co-ordinates 490 and 491.6 kb on the physicalmap of the E. coli chromosome.

7n5 irjsertion mutagenesis

Tn5 insertion mutagenesis was used to localize moreprecisely the DNA region responsible for the Hha " pheno-type. Strain E. coli 5K(pUBM22} was mutagenized withTn5tac1 and independent insertions located on the 1.7 kbEcoRV-EcoirW fragment of pUBM22. The correspondingplasmids were used to transform strain Hha-3(pANN202-312). Only two insertions (pUBM22::Tn5tac1#5 andpUBM22::Tn5tac1#6). located 85bp apart, renderedplasmid pUBM22 unable to complement the hha mutation(Fig. 2). Additionally, insertions pUBM22::Tn5tac1 A4 andpUBM22;:Tn5tac1#7. which were located only 350bpaway and flanked the former insertions, did not eliminatethe complementing activity (Fig. 2). Therefore we localizedthe hha gene to a 350bp fragment, flanked by insertionspUBM22;:Tn5tac1#4 and pUBM22:;Tn5tac1#7.

Sequence analysis of the hha gene

The DNA sequence of the region contained between Tn5insertions pUBM22;:Tn5tac1«'3 and pUBM22::Tn5-taci #7 is shown in Fig. 5. Only two open reading frames(ORFs) encoding 50 or more amino acid residues andstarting with ATG or GTG were found, both in the samestrand: ORF-1, starting at bp 87 (Val) or bp 90 (Met) and

Cloning of the hha gene from Escherichia coli 1287

9 10

i I Itt tt t t2 3 5 6 7 8

t ( 0.2 kb

Fig. 2. Tn5tac1 insertions in the EcoRV fragment of pUBM22.Numbered vertical arrows indicate the map locations of particularTnStaci insefiions. Thin honzontal arrows indicate the direction oftranscription promoted by the tac promoter of the transposon. and pointto Its O-end. Complementation of the hha mutation in the presence ofeach insertion is indicated as ( + ) or (-), and represents the reduction ofthe external haemolytic activity from average values of 15.0 units to0.004 units. Insertions * 3 . * 5 . * 6 and * 7 were used for sequencing thehha gene. Sequences to the teft of the insertion sites were obtainedusing the Tn5tact O-end primer. To sequence the complementarystrand, the plasmids to be sequenced were subjected first to EcoRIdeletion that removed the sequences from the fcoHl site located in tt>evector to the left of the insert in the Fig. (not shown) and an EcoRI site atthe l-end of the Tn5tac1. Then, using the pBR322 sequencing primer5'-GTATCACGAGGCCCT-3\ the sequences located to the right of theinsertion sites could be obtained.

ending at bp 314 (not shown in Fig. 3), and ORF-2. startingat bp 94 (Met) and ending at bp 558. It is unlikely thatORF-1 (74 amino acid residues starting frotTi Met-90)codes for Hha or is otherwise required for its expression,Tn5 insertions pUBM22;:Tn5tac1 #5 and pUBM22::Tn5-taci #6 were Hha and both are 3' to the theoretical end ofORF-1; furthermore, insertions #3 and #4 in the middle ofthis ORF were Hha *. Therefore ORF-2 is the most prob-able coding frame for hha. There are four Met residues InORF-2, between Tn5tac1 insertions * 4 and #7. at whichthe hha gene could start. They are found in positions 211.280. 343 and 379. Only Met-343 is preceded by areasonable ribosome-binding site (AGG or AGGT) atposition 333. The three best matches with the E. coliconsensus promoter are also shown in Fig. 3. The putativepromoters PI and P2 are located 5' to the Tn5 insertionpUBM22::Tn5taciF#4. Tn5tac1 should be polar in thisorientation (Chow and Berg. 1988), so the most plausiblepromoter is P3, which would start transcnption at aboutnucleotide 303. This fits well with the tact that the next Met

I TGA AAC CCT GTA TCA TGA CTG CCT TGC AAA CCT TGA AGA AAG

*3 CAA TCA TGG CTG GGT AAA CGA CCC AAC CTC GCG ATC AAC CTC

85 CAG TGA TGA ATG ATG AGC ATA TTG CGA CCT TCG CAC TTA ATT

II H

i a 7 A f A AAA TTA AGT ATA ATG AAG ACA ATA A l J f IC A T T l i ACC ACA

t l t l U3169

21 1

253

337

379

'163

505

!3'i7

599

TCG

ATGM

GTAV

1

AGAR

ATG

nGTTV

GCGA

ACCT

AAAK

CGG

ACG

GTAV

ATCI

CTG1,

AGTS

CCTR

ATC

GTAV

ATG

TTTF

CTG

AAT

TTAI.

GAC0

CGAR

ATGH*

TTA1,

GAGE

TTTF

AATN

ATTI

TTT

ATC

ATA1

TATY

GTTV

TCCS

CGTR

AAAK

TACf

AAAK

CGCR

ATT

TGG

TGCC

TCC

aTATV

GAA

CGTR

AATN

TCAS

CTG1.

TAAZ

AAG

ATG

AGGR

V

I'TT1.

AAAK

TGCr

AAAK

RCCA

TACy

TAA7.

AAT

ACA

ATCI

{>

GTTV

11

GTTV

P

CAG

TACY

GCAA

GAC

ATA

TTT

iCCT

TTCK

ITG1.

AGAR

TTA1.

ACAT

GAAF

GATD

AAGK

ATT

ATA

TTA

AGAR

TCAS

ATT

ACi;T

ATT1

TTA1,

CJl|c

K

ATCI

CGC

CTT

TGT|

AATN

ATGH

ATT

AAAK

GACU

TGT

GGCG

CGAR

ACAT

ACC•I'

Acjt;T

T C A G A T.1 t>ttl

CGCR

CCTP

TTT

TTT

CTCL

TCCS

CGG

CGC

TCA

GGAG

CGAR

ACCT

GTA

AGTS

GTT

CAGQ

AAG AGA

I*-—ft-'

GAT TATT> YmCTGL

AATN

GtjcA

TCAS

ACC

CAT

GAGE

GAAK

GAAE

GTAV

TAT

GAA

TTAI

CC|TR

CTGL

TTGL

TGGN

AAC

GAC

310

252

336

378

(tio

462

50*

546

5sa

630

Fig. 3. Nucteotide sequence of a 707 bp Iragnwntcontaining the hha gene, and deduced ammoacid sequence ot the Hha protein. P I , P2. and P3are the putative promoters located in thefragment. Arrows below the sequence indicatethe two most plausible translation stan sites(Met-211 and Met'343). Asterisks indicate thetranslational stop codons. The Shine-Oalgarrx)sequence preceding Met-343 is shown by adotted box. Target sequences duplicated in thodifterent Tn5tac1 insertions are boxed All lourinsertions were used for sequencing the fragmentas explained in the legerxl to f ig. 2. Thesesequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries underthe accessk>n number X57977 {E. coli hha).

631 ATA CCC TAT GTG ATC ITT ATC ACA t.AC ATI! TAA TGC UAA C(;T

ttf673 TCT CTT CAC TGA CTT TTC GTC TTA ATIG TtJT TGC Clj 7 0 7

6 7 2

1288 J. M. Nieto eta\.

kDa

46.0 -

30.0 -

14.3 -

Fig. 4. Electrophoretic analysis of proteins expressed by recombinantplasmids in a minicell system. Samples were run on an acrylamide-ureagel as described by Anderson et al. (1983). Lane A, plasmid pUBM22;lane 8. plasmid pUBU23: lane C, plasmid pBR322: lane D. molecularmass marker. The arrow points to the polypeptide expressed in ptasmidspUBM22 and pUBM23. The poorly expressed low-M, polypepttdedetected in minicells harbouring pBR322 plasmid probably correspondsto the rop gene product, a 6500Da protein (Cesarini el a/., 1982).

is Met-343 (the one which is preceded by the Shine-Dal-garno sequence). We assume, theretore. that Hha is a72-amino-acid protein starting at Met-343 with a predic-ted M, of 8629 Da. An abundance of charged amino acidssuggests a very hydrophilic soluble protein, as can bejudged by its hydropathic profile.

We also examined the Hha amino acid sequence forhomology with other E. coti or bacterial proteins, by usingthe GCG sequence analysis software package. Thehighest percentage of identity (29%) was detected for a31 -amino-acId overlap with the SulA protein (FreudI e( ai.1987), corresponding to the amino-terminal region of theHha protein. Also. 23% identity was obtained for the wholeHha sequence with the recB gene that codes for Exonu-clease V, a 133973Da polypeptide (Finch et al., 1986).Additionally, the Hha polypeptide was searched forregions similar to the helix-turn-hellx tertiary structurecommon to several DNA-binding proteins (Alba et al.,1982). This structural motif, characterized by a glycineresidue flanked by hydrophobic amino acids fourpositions before and six positions after the glycine (Paboand Sauer, 1984), was not apparent for the Hha protein.

Minicell analysis

To identify the expressed Hha protein, polypeptidesencoded by plasmid pUBM22 were expressed in minicells(Fig. 4). It could be assumed from insertional mutagenesisdata, as well as from the sequence data, that the Hhaprotein was a low-M, protein. Therefore, we analysedminicel! extracts by polyacrylamide gel electrophoresis(PAGE) as described by Anderson and co-workers (Ander-son e( al, 1983) in order to separate low-M, polypeptides.

Upon autoradiography, extracts containing pUBM22showed a protein of about 8000Da (Fig. 4). This resultreinforced our assumption that the ORF for hha starts atMet-343. which would result in a protein of M, 8629Da.The protein starting at Met-211 would result in a polypep-tide of 13 765 Da. which is too large for the band observed.However, the difficulty in evaluating the molecular mass ofsmalt proteins from sodium dodecyl sulphate (SDS)-PAGEmigration data should be stressed. Minicell extracts ofstrains containing the Hha" plasmids pUBM22::Tn5-taci #5 (Fig. 5) and pUBM22::Tn5tac1 #6 (not shown) didnot show the 8000 Da band. In contrast, minicell extractsfrom clones harbouring the Hha*̂ plasmids pUBM22::Tn5-tac1#4 (Fig. 5) and pUBM22::Tn5tac1#7 (not shown)showed the identified polypeptide.

Effect of the hha mutation on haemolysin expression

To obtain further information about how the hha mutationmodulates haemolysin expression, we used Westemblotting to evaluate the overall amount of haemolysinpresent in the different cellular compartments of both E.coll 5K and E. coli Hha-2 harbouring the haemolyticplasmid pANN202-312. The 110kDa polypeptide corres-ponding to haemoiysin could not be detected in thesupernatant of cultures from strain E coli 5K(pANN202-312). In contrast, Hha-2 cells harbouring plasmid pAN-N202-312 showed the 110 kDa polypeptide (Fig. 6). Inaddition. 5K cells harbouring a ptasmid including hlyRshowed the HlyA protein in the culture supernatant. Inaddition, densitometric analysis of Western blots showedthat the hha mutant contained approximately four timesmore haemolysin in the cytopiasmic compartment than

kOa

46.0-

30.0-

14.3-

A B C

Fig. 5. Electrophoretic analysis of proteins expressed in minicellsharbouring plasmids pUBM22::Tn5tac1 #4 and pUBM22;:Tn5taC(»'5.Electrophoretic condilions were as described in Fig. 6. Lane A.molecular mass marker; lane B, plasmid pUBM22::Tn5taQi if 4; lane C,plasmid pUBM22::Tn5tac1 ifS.

Cloning of the hha gene from Escherichia coli 1289

A B C

Fig. 6. Western blot analysis ot tt>e cuKure supernatants from strains5K{pANN202-312) (lane A). 5K(pANN202-3t2R) (lane B) and Hha-2(pANN202-312) (lane C). The arrow points to the 110-kDa HlyApolypeptide.

did the parental 5K strain {data not shown). These resultssuggested that cytoplasmic synthesis of haemotysin maybe increased in the mutant strain or that intracellularhaemotysin has higher stability. To obtain further informa-tion we used Northern analysis to measure the level ofhaemotysin-specific mRNA in both the parental andmutant strains. The major hlyCA transcript could bedetected in preparations of both 5K(pANN202-312) andHha-2(pANN202-312) strains, indicating significantincrease of that transcript in the mutant strain. Theseresults correlate well with the previous observation thatthe Hha-2 strain contains significantly higher amounts ofintracellular haemolysin than the parental 5K strain. Inspite of the important increase in the levels of the hlyCAtranscript in strain Hha-2(pANN202-312), the minor 8kbh/yC>ASD transcript could not be detected (Fig. 7). Never-theless, the hlyCABD transcript was detected in extractsof the Hha-2 mutant strain when cells harboured plasmidpANN202-312R. which contains the Wyf? sequence pre-ceding the haemolysin determinant {Fig. 7).

that minicell analysis showed a protein band of approxi-mately 8000 Da.

With respect to the mechanism of action of the Hhaprotein, there are a number of results available whichseem relevant to this question. We show here that strainsof E. CO//containing the Hha mutation show a significantincrease in concentration of extracellular as well ascytoplasmic haemolysin, as determined by immunoblot-ting, thus suggesting that the hha mutation increasessynthesis of the HlyA protein. This effect resembles that ofhlyR. a poorly characterized activating sequence locatedupstream of the hly operon (Gonzalez-Carrerd e( al., 1985;Vogel et al., 1988). tn this respect, E. coli Hha-2 carryingplasmid pANN202-312R {containing htyR) has an externalhaemolytic activity twice that of otherwise isogenic hha'cells (Godessart etal.. 1988). Therefore, the mechanism bywhich the hha mutation increases haemotysin synthesis isquantitatively different. It has been proposed that the maineffect of hlyR is to avoid temiination in the hlyCABDoperon, thus increasing transcription of the htyB gene(90-fold increase), although a slight increase in hlyAtranscription (three-fold) was also detected (Koronakis etal.. 1988). In contrast, the main effect of the hha mutationappears to be to increase the levels of the hlyCA transcript.We have been unable to detect the /7/>'C/̂ SD transcript inhha cells harbouring plasmid pANN202-312. The htyRsequence appears also to be needed in the hha geneticbackground for expression of the hlyCABD transcript atdetectable levels. These results reinforce the assumptionthat the hha mutation and the hlyR sequence modulatehaemolysin expression by a different mechanism. Never-theless, the possibility that hha mutation also causes aslight increase in transcription of the hlyB and hlyD genes.

Discussion

In this study we have been able to localize the hhamutation to a single gene, which has been ctoned andsequenced. This allowed us to establish that the remark-able increase in haemolytic activity in E coti cells harbour-ing the recombinant haemolytic plasmid pANN202-312and the hha mutant allete was the result of inactivation ofthe Hha protein. Analysis of the sequence revealed twoORFs (ORF-1 and ORF-2) encoding proteins of 50 or moreamino acid residues starting with ATG or GTG codons.The onty likely coding frame for hha is ORF-2. as deducedby the effect of the different Tn5tac1 insertions on the Hhaphenotype {see Fig. 5). Two possible polypeptides of13765Da and 8629Da, starting at Met-211 or Met-343.respectively, could be synthesized from ORF-2. We pro-pose that Met-343 is the most likely translational startpointfor Hha. This assumption stems from two considerations:(i) that only the polypeptide starting at Met-343 is pre-ceded by a reasonable Shine-Dalgarno sequence, and {ii)

hlyCABD

hlyCA

Fig. 7. Northem analysis of haemolysin specific mRNA. Total celluivRNA (20ng) from strains 5K(pANN202-312) (lane A). Hha-2(pANN202-312) {lane B) and Hha-2(pANN202-312R) (lane C) was fractionated byagarose gel electrophoresis. Upon blotting to nitrocellulose, the RNAwas hybridized to a ^P-radiolabelled probe corresponding to hlyA (seethe Experirrwnlalprocedures). The arrows pornt to the 4,0kb hlyCA and8.0 kb hlyCABO transcripts. Unlabelled arrowheads indicate bandscorresponding to 23S and I6S RNA, an artifact usually present dunnganalysis ot prokaryotic RNA by Northern blotting (Welch and Pellet.1986).

1290 J. M. Nieto etal

perhaps because of readthrough between hlyA and hlyB,cannot be ruled out. Such an effect would justify anincrease in HlyA secretion, which in turn could alsoinfluence HlyA expression if, as seems likely. HlyA transla-tion and secretion are coupled to some degree. Anincrease in HlyA secretion would release a hypotheticalindirect repression of HlyA translation and also of hlyAtranscription if both are coupled. Elevated levels of htyCAmRNA (and of internal HlyA protein) in Hha mutants canbe due either to an increase in the transcription rate or tohigher stability of the transcript. As the overall effect of theHha protein is to decrease the expression of the Wy genes,this protein might be involved, either directly or indirectly,in modulation of transcription or. alternatively, in mRNAturnover. A homology search did not reveal functionallysimitar proteins. The closest matches (see the Results)associated the Hha protein erther with SulA (Freudl e( a/.,1987) or with Exonuclease V (Finch ef a/.. 1986). From thededuced amino acid sequence a 24.3% identity wasfound with the Cyt repressor (Valentin-Hansen et al.,1986). The significance of such homologies. if any, cannotbe assessed at this stage. Tlie Hha protein lacks theconsensus sequence established for binding domains ofDNA-binding proteins and therefore is unlikely to actdirectly as a transcriptional regulator. In addition, nohomology could be detected between Hha and histone-like proteins of bacteria, also known to modulate tran-scription in various systems (Driica and Rouviere-Yaniv.1987).

With respect to mRNA turnover, conclusive biochemicaldata are not yet available. The predicted Hha amino acidsequence is not significantly related to any reportedbactenal RNase. Nevertheless, the processing andbreakdown of bacterial mRNA is still poorly understood.We cannot rule out the possibility that the observedincrease in the overall amount of haemolysin-specificmRNA in E. co// Hha is due to the enhanced stability of thehlyCA transcript, it is remarkable that defined processeswhich involve cleavage of mRNA have still not beenassociated with specific proteins (Nilsson et al. 1988;Meader, III, etal., 1990).

Clarification of the precise mechanism by which the Hhaprotein modulates haemolysin expression requires furtherexperimental data. Purification of the protein is proceed-ing in our laboratories, and we believe that this work willhelp to define the biochemical activity of this apparentlynovel modulator of gene expression in £ coli.

Experimental procedures

Bacterial strains, piasmids and phages

The strains, recombinant piasmids and phages used in thepresent study are listed in Table 1.

Media and genetic manipulations

LB medium (Miller, 1972) and blood agar (Godessart et al.. 1988)were routinely used for the maintenance and growth of the strains.When required, ampicillin, tetracycline. chloramphenicot or kana-mycin were added at the concentrations indicated elsewhere{Maniatis etal.. 1982). Plasmid DNA isolation, transformation andrestriction analysis were performed as previously described(Godessart ef al., 1988). Chromosomal DNA was isolated aspreviously reported (Silhavy et al., 1984).

Protein analysis

Fractionation of proteins on 11 to 18% SDS-polyacrylamide gelswas carried out by a modification (Ames ef al., 1973) of themethod of Laemmli (Laemmii. 1970). Separation of low-M,polypeptides was performed by following the method of Ander-son (Anderson ef al., 1983). Silver staining of acrylamide gels wasperformed as previously described (Merril ef al., 1984).

Haemolysin assay

Haemolytic activity was assayed as previously described (God-essart eta/.. 1988).

Cloning of the hha gene

Gene hha was cloned In vivo by using the mini-Mu replicon Mud005 (Groisman and Casadaban. 1986). To do this, strain E. coliC600 (Mu cts) was transformed with plasmid pEG5005 andthermoinduced as descnbed (Groisman and Casadaban. 1986).The lysate obtained was used to infect E. coli Hha-2(pANN202-312). After plating on blood agar plates containing ampicillin,chloramphenicol, and kanamycin, bacterial colonies comple-menting the hha mutation were isolated and plasmid DNA purifiedfrom them for further characterization.

Cloning of an BcoRI fragment, including the Km mailer,from strain E. coli Hha-3

To clone the Tn5-derived Km" gene from strain E. coll Hha-3.chromosomal DNA was isolated, digested with EcoRI, andcloned into pBR322. Upon transformation in strain E coli 5K.selection was pertormed on L6-kanamycin plates.

Insertional mutagenesis with Tn5

Transpositions to plasmid pUBM22 were obtained by infectingstrain 5K(pUBM22) with X::Tn5tac1 (Chow and Berg. 1988).Plasmid DNA was extracted from pools of Km" transductants andused to transform strain E. coli DH1 to Km". Insertions intopUBM22 were mapped by restriction analysis and the comple-menting activity was tested transforming the piasmids containingthe insertions into strain Hha-3(pANN202-312).

Nucleotide sequence analysis

DNA sequences were determined by the chain terminationmethod, using plasmid DNA (purified by CsCl/ethidium bromide

Cloning of the hha gene from Escherichia coli 1291

Table 1. £ coli strains, phages and piasmids.Strain/Plasmid/Phage

Strak)

SK

[>S4tO

DH1

CfiOOMucfs

Hha2HhaS

Plasmid

pHly152pANN202-312

PANN202-312RpEG500S

PUBMS005-1 toPUBM500S-5

pUBMZOpUBM21PUBM22PUBM23pU8M22::Tn51acl#1.

»'2. #3. * 6 . * 7 t opUBM22::Tn5tac1#11pUBM22::Tn5tac1#4and #5

Rwge

Lambda: :Tn5tac1

Relevant genotype/Phenotype

F , hsdR, h$dM, rpsL thr, tN, hu.IacZ

F . minA. minB. thi. mtt. ara. xyl.gal, tonA. rpsL

F . racA 1, endA J. gyrA96, thi-1.hsdRI7.supE44. relAI

thr. leuB6. thi. supE44. lacYI.fhuA2l Mu cts

SKhha:.Mud^5K hha::Tn5phoA

hlyR. hlyC. hlyA. hlyB. hlyDhtyC. hlyA hlyB, NyD. Cm"

hfyfi, hfyC. htyA. hlyB, htyO. Cm"p8C0:;Mu d5005

hha •. Km"hha•. Ap"Mia •. Tc"Wia ' .Ap"Ww .Ap"Ww• .Ap^Km' '

hha'. Ap", Km"

Ap^.Km"

Km"

Source/Reference

Godessanefsf. (1968)

Dougan and Sherratt(1977)

Hanahan(1983)

Groissman andCasadaban (1986)

Godessad eraf. {1988)Godessaneraf. (1988)

Ncwoai«(ltf.(1981)Gosbel arid Hedgpeth

(19a?)Godossan ei a/. (1988)Groissman and

Casadaban (1986)

This studyThis studyThis studyThts studyThis studyThis study

This study

This study

Chow and Berg11988)

density gradient centrifugation) as a template (Korneluk et al.,

1985). Ttie primers used were 5'-CCCCTACTTGTGTATAAG-3'

(from ttie Tn5tac l O-end). and 5'-GTATCACGAGGCCCT-3'

(trom ttie pBR322 vector). (See the legend of Fig. 2 for further

details.)

bovine serum albumin for 60 mm at room temperature, fitters weretransferred to a solution containing rabbit polyclonal antiserumraised against purified HtyA protein. Detection of the haemolysirv-antibody complex was [jerformed wrth anti-rabbit IgG alkalinephosphatase conjugate (Boehringer Mannheim).

Expression in minicells of the recombinant DNA

Minicell-producing strain DS410 was transfornied with plasmidDNA. minicells were purified, and plasmid-encoded proteins werelabelled with p^S]-methionine as described (Stoker ef al.. 1984).Lataelled proteins were analysed by SDS-PAG6. either followingthe method of Laemmli or the method of Anderson for low-M,proteins. Autoradiography allowed detection of the labelledpolypeptides upon electrophoresis.

Westem blotting

The expression of HlyA was monitored by Western blotting, Cellswere grown in LB medium to the mid-log phase, collected bycentrifugation, and fractionated as descnbed (Nieto et a/.. 1987).Proteins present in the cytoplasm, periplasm and supernatantwere separated by etectrophorests through 8% polyacrylamidegels (10% SDS) and transferred to nitrocellulose membranes byelectroblotting. Upon blocking of residual protein-binding sites byincubation in 0.05 M Tris-buffered saline (pH 7.5) containing 5%

RNA blotting

Total cellular RNA trom strains 5K(pANN202-312). Hha-2(pAN-N202-312) and Hha-2(pANN202-312R) was prepared as pre-viously descnbed (Mellado et ai, 1981). The R f ^ wasresuspended in sample buffer (500^1 ot deionized formamide,lOOfil of lOx MOPS, 160(il of 37% formaldehyde, 30jil ofgiycerol, lOjxl of bromophenol blue) and identical amounts foreach strain were fractionated by agarose gel electrophoresis asdescribed (Maniatis et al.. 1982). RNA was transferred to anitrocellulose membrane (Mithpore) by blotting (Maniatis ef al.,1982). For the detection ot haemolysin-specific transcnpts wegenerated a radiolabelled probe as follows. A 793-bp tcoRI-H;hdtll tragment corresponding to hlyA was cloned into plasmidpSP65 (Melton etal., 1984). A strand-specitic ^P-labelled RNAprobe was generated in vitro with SP6 potymerase. RNA washytwidized to blots ovemight in 40% formamide. 5 y Denhardt's.50mM NaPO^. pH 7.0. 5mM ethyienediamine tetraacetic acid(EDTA) pH 8.0. 0.2% SDS. 150ng ml ' salmon-sperm DNA and5x SSC. The blots were washed first in 2x SSC/0.2% SDS.

1292 J.M. Nieto etal

afterwards in l x SSC/0.1% SOS at 65°C, and dried andsubjected to autoradiographv.

Acknowledgements

This work was supported by the CICYT (Grant BIO88-0407-CO2)to F.C. and A.J. J.N.. Y.J. and S.B. were recipients of P.F.P.I.fellowships from the Spanish Ministry of Education. M.C. wasrecipient of a F.I. fellowship from the Generalitat de Catalunya.The authors thank M. Casadaban for plasmid pEG5005 and strainC600 Mu cts. Matilde Cabezas for technical help in sequencingthe hha gene, and Maria Vilanova for help with the computersearch for ONA sequence and amino acid sequence homologies.

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