Gene, 96 (1990) 141-145 Elsevier

141

GENE 02816

H U - I mutants of Escherichia coli deficient in D N A binding (Recombinant DNA; histone-like protein; sito-directed mutagonesis; hupA-hupB and himA mutants; mini-F plasmid; RSF1010; Mu phage)

Naoki Goshima, Kyoko Kohno, Fumio Imamoto and Yasunobu Kano Department of Molecular Genetics,Institute of Molecular and CellularBiologyfor PharmaceuticalSciences, Kyoto Pharmaceutical University,

Yamashina-ku,Kyoto (Japan) Received by A.J. Podhajska: 24 July 1990 Accepted: 20 August 1990

SUMMARY

We constructed four mutants of the Escherichia coli hupB gone, encoding HU- 1 protein, by synthetic oligodeoxyribor,,ucleotide-directed, site-specific mutagenesis on M 13mp18 vectors. The HupBR45 protein contained alterations of ArgSS-, Gly and Arg61 -, Gly, and the HupBF3, HupBK2 and HupBAI proteins contained Phe47 -, Thr, Lys37 -, Gin and Ala3° --, Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at a2°C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBAI and HupBK2 supported these cellular activities. DNA-alTmity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the ann structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short//-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functicns of this protein.

INTRODUCTION

The HU-like proteins are evolutionarily well conserved in prokaryotos. In E. coli, HU consists of two molecular species, HU-I and HU-2, with highly homologous aa sequences and constitutes a major fraction of DNA-binding proteins associated with nucleoids (for reviews, see Drlica and Rouvi~re-Yaniv~ 1987; Pettijohn, 1988). The hupB and hupA genes, encoding HU-1 and HU-2, respectively, have been cloned and mapped (Kano et al., 1985; 1986; 1987; Corresp,~ndenceto: Dr. Y. Kano, Department of Molecular Genetics, Institute of Molecular and Cellular Biologyfor Pharmaceutical Sciences, Kyoto Pharmaceutical University, 1, Shicbonocho, Misasagi, Yamashina-ku, Kyoto, 607 (Japan) Te1.(81-075)593-2920; Fax(81-075) 502-1613. Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); CBB, Coomassie brilliant :due; Cm, chloramphenicol; A, deletion; DTT, dithiothreitol; EtdBr, ethidium bromide; Hopes, N-(2-hydroxyethyl)0378-1119/90/$03.50 © !990ElsevierSciencePublishersB.V.(BiomedicalDivision)

1988). The hupA-hupB double deletion mutants cannot support o~2-dependent DNA replication of the mini-F plasmid (Wada et al., 1988b), replicative transposition of Mu phage (Kano otal., 1989), or site-specific DNA inv~,rs;on of bin, gin, pin and shuffion systems (Wada et al., 1989L The latter two are consistent with previous in vitro resvi:s (Craigie et al., 1985; Johnson et al., 1986). A current idea is that HU serves as an accessory factor in facilitating other protein-DNA interactions by bending the DNA and bringing separated signals close (Hodges-Garcia et al., 1989). piperazine-N'-2-ethanesulfonic acid; HU, see INTRODUCTION; hupA, gene encoding HU-2; hupB,gene encoding HU-I; IHF, integration host factor; kb, kilobase(s) or 1000 bp; Km, kanamycin; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide;PA, polyacrylamide; PAGE, PA-gelelectrophoresis; R,resistance/resistant; SDS, sodium dod~cylsulfate; Sin, streptomycin; Tc, tetracycline; ts, temperature sensitive; wt, wild type; [ ] denotes plasmid-carrier state; ::, novel joint (fusion).

!42 HU protein forms an arm structure consisting of a double stranded antiparallel fl-pleated sheet in the C-terminal region (Tanaka et ai., 1984). The arm region contains several Arg residues that have been implicated in D N A binding (Lammi et al., 1984). In the N-terminal half, a V-shaped structure composed of two a-helices, has been speculated to be the domain for dimer or oligomer formation (Tanaka et al., 1984). The aim of the present study was to assess the nature of H U - D N A interaction and the precise role of this factor in biological reactions by constructing point mutations of the HU-I protein that alter the ability of DNA-binding.

hupB

#upBA1

Sequence 30 Leu Asp Ala lie lie Ala Ser Val Thr Glu Ser TTA G6,TGOTATTAT[ GOTTCC GTAACT ~ TOT Leu Asp Ala lie

Asp Set Val Thr Glu Ser

"l'rA GAT GCT ATT A.T.CGAT TCC GTA ACT GAA TOT CloI 37 Val Thr Glu Set Leu Lys Glu Giy Asp Asp V¢; GTA ACT GAA TCT CTG AM GAA GGGGAT GAT GTA

Val Thr Glu Ser Leu Gill Giu Gly Asp Asp Val GTA ACT GAA TCT CTG GACGAA GGGGAT GAT GTA PstI 47 Val Ala Leu Val Gly Phe Gly Thr Phe Aia Val GTA GCA CTG GTA GGT TTr GGT ACT TrT GCOGTT

EXPERIMENTAL AND DISCUSSION (a) Construction of point mutants in H U . I protein Using synthetic oligo-directed, site-specific mutagenesis of the hupB gene on M13mpl8 vector, we obtained four mutations. One (hupBR45) contained two alterations in HU-1 protein, ArgSa-,Gly and Arg61-,Gly, and the others contained a single alteration of Phe47--,Thr (hupBF3), Lys 37 -, Gin (hupBK2) or Ala 3°--, Asp (hupBA1). The n~.changes associated with the replacements of these aa residues are shown in Fig. I. The changes in the nt sequence were designed to create a new cleavage site for the unique restriction enzyme in a 1664-bp BamHIHindIII DNA fragment containing the hupB structural gene (Fig. 1). Creation of the site was shown by the production of fragments ofreasonable size by complete digestion of the phage DNA with the restriction enzyme (Fig. 2). Sequencing confirmed that each mutated hupB gene contained only the mutation introduced by the synthetic oligo (data not shown). (b) Effects of mutations on HU-production and cell growth The mutated HU-1 proteins were characterized by introducing the phupB plasmid (mutated hupB-carrying pBR 322) into a strain YK 1340 which deleted both the hupA and hupB genes. On S D S - P A G E of partially purified proteins from these transformants, the HU-1 proteins synthesized from phupB plasmids comigrated with wt HU-1 protein synthesized from pMW1 (Fig. 3A). The levels of the protein in this position were comparable to that from pMW1 (Fig. 3A). The band of HupBR45 on the Westernblot filter was visible but not as strong as the others (Fig. 3B), suggesting that HupBR45 did not react with the antibodies as strong as other HU proteins, possibly due to alteration in its configuration.

The hupA-hupB double-deletion mutants grow normally at both 37°C and 42°C in LB broth, but cells harboring an additional mutation of the himA gene exhibit ts growth at 42°C (Kano and Imamoto, 1990). This observation indi-

Val Ala Leu Val Gly Thr Gly Thr Phe Ala Val GTA GCA CTG GTA 8GT ACG GGT ACT TTT GCOGTT

hupBR45

KpnI 58 61 Arg Ala Ala Arg Thr Gly Arg Asn Pro Gin Thr CGT GOT GCOCGT ACT GGCCGOAAO CCGCABACC Arg Ala A l a ; ~ ?:~ Gly Gly Ash Pro Gin Thr CGT GCI~~CG GGCACT GGCGGCAAO COGgAG ACC Nod

Fig, !. The nt and aa sequences of mutated parts of the hupB gene. A Muta-Gene in vitro mutagenesis kit (Bio-Rad) was used for mutagenesis of hupB gene. Mutagenic oligos were assembled on an .Applied Biosystems 381Aautomated DNA synthesis apparatus by the 2-cyanoethylphosphoramidite method and purified on an OP,~. column (Applied Biosystems,Inc.). The BamHI-Clal fragrrent containing wt hupBand its promoter was prepared from pMWl which carries wt hupB gene (Kano et al., 1986). The fragment was cloned into BamHI + Accl-digested M13mp18and site-directed mutagenesisof the hupBgene was carried out according to Kunkel et al. (1987). The followingsynthetic primers were used to direct the mutations; 5'-AGTTACGGAATCGATAATAGC. ATC-3' for hupBAl (Ala3°-~Asp); 5'-TCATCCCCTTCCTGCAGA. GATTCA-3' for hupBK2 (Lys37-~ Gin); 5'-GCAAAAGTACCGGTACCTACCAGT-3' for hupBF3 (Phe''7 ~ Thr), and 5'-CGGGTTGCCGCCAGTGCCGGCAGCACGCTCTTT-3' for hupBR45 (ArgSS--~Gly and Arg6' ~ Gly). The changes in the nt sequence were designed to create a new cleavage site for the unique restriction enzyme in the hupB coding regions: a Clal site in hupBAl, Pstl site in hgpBK2, Kpnl site in hupBF3, and NaeI site in hupBR45. The upper a:a and nt sequences represent part of the 'at hupB; the mutant hupB s~quences are shown below. The aa residues are numbered from the N-terminal aa (= 1). The mutated aa of HU-I are underlined. The nt changes are indicated by arrows. New restriction sites are doubly underlined.

cares the requirement of HU protein for growth of E. cob in the absence of IHF, suggesting functional similarity betwcen HU and IHF. We, therefore, examined the effect of the mutated HU-1 proteins on cell growth in a hupAhupB-himA triple mutant (YK2741). Unlike control cells with pBR322, the transformants of YK2741 with

143

Clal 1 2

Pst I 34

Kpn I 56

Nae I 78 ~-7~138~ "4

~'~" q--6841

!

.-1368

Fig. 2. Identification of nt changes in the mutated hupB fragments. Lanes 1, 3, 5 and 7 contain DNA from the recombinant M13mpl8 phage with the wt hupB gen¢. Lanes 2, 4, 6 and 8 contain recombinant M 13mp18 with hupBA1, hupBK2, hupBF3 and hu,~BR45, respectively. Phage DNAs were digested with Cial (lanes I and 2), Pstl (lanes 3 and 4), Kpnl (lanes 5 and 6) or NaeI (lanes 7 and 8), and run on 1% agarose gel, except lanes 3 and 4 which were run on 8% PA gel in TBE buffer (90 mM Tris/90 mM borate/2 mM Na2" EDTA pH 8) and stained with EtdBr. The DNA fragments identified are indicated by arrows. Numbers represent molecular sizes in bp of the DNA fragments.

phupBR45 or phupBF3 did not show detectable growth at 42 ° C on LB agar plates containing Ap (Table I). The viable cell number was less than 10 -4 of that at 37°C. The transformants with phupBA1 or phupBK2 exhibited similar growth characteristics at 37°C and 42°C as the cells with pMW1.

(c) Effects on mini-F and RSFIOIO replication and Mu phage development Mini-F plasmids are not stably maintained in hupA-hupB double-deletion mutants because of the deficiency of or/2-dependent DNA replication in the absence of HU protein. These mini-F plasmids could replicate in hupsingle

A

o '1

,iiiiit CBB

Anti- HU

Fig. 3. Electrophoretic analysis ofmutated HU-I proteins. The BamHI + HindlIl.digested fragment carrying mutated hupB from the recombinant phage Ml3mpl8 as described in the legend to Fig. 1 was re-cloned into BamHI + HindIII-digested pBR322 to construct the pbupB plasmid. The phupBAl, phuBK2, phupBF3 and phupBR45 plasmids thus obtained carry mutations hupBA1, hupBK2~hupBF3 and hupBR45, respectively. These mutations were confirmed by DNA sequencing. (Panel A) The hupA-hupB double-deletionmutants (YKI340), harboring mutated phupB plasmids listed above the lanes, were harvested, suspended in 200 #1 of SDS sample buffer (65 mM Tris. HCI pH 6.8/3% SDS/10% glycerol/100 mM DTr), and sonicated. The homogenates were heated at 100°C for 2 min and loaded onto 0.1% SDS-15-18% PA gel (Laemmli, 1970). Proteins were stained with CBB and then the gels were destained by diffusion. (Panel B) For immunoblotting analyses, the proteins fractionated as in panel A were transferred to a nitrocellulose membrane with a Saltblot II semi-dry blotter (Sartorius). The blots were treated .with mouse ~n.ti-HU antiserum and HU proteins were detected with alkaline phosphatase-conjugated antimouse-immunoglobulin.

144 ~ABLE I

TABLE III

Colony formation by transformed hupA-hupB-himAmutants "~

Effec~ of mut.ated HU-I proteins of efficiencyof Mu plaque formation

Plasmidb

37°C

42°C ¢

pBR322 pMWI phupBA1 phupBK2 phupBF3 phupBR45

+ + + + + +

+ + + -

~' E. coli K-12 triple mutant YK2741, hupAl6(KmR) hupBll(CmR) A82[himA]::Tn10derivative ofYK1100 (Kano and Imamoto, 1990)was transformed with plasmids listed, and an ApR colony was selected on LB plate (LB broth containing 1.5% agar) containing 50/ag Ap/ml at 37°C. The transformant.~were grown at the indicated temperature on LB plates containing 50/ag Ap/ml. hupAl6 and hupBll are insertion-deletion mutants ofhupgenes in whichhupAis replaced by Kmg and hupBby Cm~ fragments, respectively. b See legends to Figs. 1 and 3 for pMWI and other plasmids, respectively. Symbols + and - at 42°C indicate that the plating efficiencies of bacteria were, respectively, similar to and < 10-4 those at 37°C.

mutants producing either HU-1 or H U - 2 (Wada et al., 1988b). We, then, examined the ability of mini-F plasmid replication in the YK 1340 (hupA-hupB) [phupB ]. As shown ir Table II, the mini-F plasmid p F T C 1 transformed the hup double mutant harboring p M W l but not pBR322. Introduction of phupBA1 or p h u p B K 2 overcame the deficiency

TABLE II Transformation of the hupA-hupBmutant with mini-F plasmid pFTCI or plasmid RSFIOIO Plasmid~

pBR322 pMWl phupBA1 phupBK2 phupBr:3 phupBR45

TcR transformants

SmR transformants

Obtained b

Ratio d

Obtained c

Ratio d

0 67 153 194 0 0