Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli

J. Mol. Biol. (1988) 204, 581-591

Construction and Characterization of the Deletion Mutant of hzqA and hupB Genes in Escherichia coli M. Wada’, Y. Kane’, T. Ogawa2, T. Okazaki2 and F. Imamoto’ ‘Laborato r y of Molecular Genetics (Riken) Tsukuba Life Science Center The Institute of Physical and Chemical Research, 3-l -1- Iioyadai Tsukuba, Ibaraki 305, Japan ‘Department School

of Molecular

of Science,

Biology

Nagoya University

Nagoya 464, Japan (Received 26 April

1988)

Insertion and deletion mutations of the hupB and hupA genes, which encode the KU-1 and HU-2 proteins, respectively, of Esch,erichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome. The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively. A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene. Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain. The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 “C. Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress. The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

have also mapped these genes: hupB is at 9.7 minutes (Kano et al., 1986), and hupA is at 90.5 minutes (to be published), on the &scherichia coli chromosome. The HU protein is a small, basic protein and is a major component of the proteins bound in the E. coli nucleoid (Rouviere-Yaniv, 1978; Yamazaki et aE., 1984). It is most likely that native HU protein is a heterodimer (Rouviere-Y aniv & K jeldgaard, 1979) of two highly homologous components, HU-1 and HU-2 (Mende et al., 1978; Laine et al., 1980). The HU protein binds to doublestranded DNA (Berthold & Geider, 1976) and have been shown to retain negative supercoils in DNA (Rouviere-Yaniv et al., 1979; Broyles & Pettijohn, 1986). HU protein exhibits several additional functions in vitro: it is involved in DNA replication from E. coli oriC (Dixon & Kornberg, 1984; Ogawa et al., 1985), in transposition of Mu phage (Craigie et al., 1985) and in site-specific inversion of the H

1. Introduction Bacterial DNA is organized into a higher-order structure by association with DNA binding proteins such as the HU proteins (Rouviere-Yaniv & Gross, 1975; Griffith, 1976; Varshavsky et al., 1977; Rouviere-Yaniv, 1978; Yamazaki et al., 1984). The molecular basis of formation and maintenance of such a structure, called a nucleoid (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972; Kornberg et al., 1974; Champoux, 1978), is not understood. However, it seems reasonable to assume that nucleoid structure is important for such cellular functions as gene expression, DNA replication, DNA inversion and DNA recombination. To approach an understanding of the physiological significance of nucleoid structure and to facilitate further studies on the biological function of HU protein, we have cloned and sequenced both the hupB and hupA genes (Kano et al., 1985, 1987). We 581 0022-2836/88/230581--l

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region of’ Salmonella typhimurium (Johnson et al.. 1986). These observations suggest that HU protein plays an important role in a number of fundamental cellular functions. In this paper, we report on the construction and physiological characterization of mutants carrying deletions of the hupB and hupA genes.

2. Materials and Methods (a) Bacterial strains and plasmids

The E. coli strains and plasmids used are listed in Table 1. Transformation of E. coli strains with plasmids was performed as described (Mandel & Higa, 1970). After transfection of DNA in 0.1 M-Ca&, cells were incubated in L-broth (1 o/o Bacto tryptone: 0.5% Bacto yeast extract, 0.5% NaCl, 0.1 o/o glucose) at 37 “C! for 60 min. The transformants were selected at 37°C on L-broth containing 1.5% Bacto agar and 25 pg plates or 50 pg ampicillin/ml, 15PLg chloramphenicol/ml kanamycin/ml. Genetic crosses were performed by bacteriophage Plkc-mediated generalized t’ransduction (Miller, 1972).

(b) DNA methods Cloning procedures. including restriction rndonuclease digestion. ligation, gel electrophoresis and Southern blotting were performed according to Maniatis rt al. (1982). Labeling of restriction fragments with 32P-labeled nucleotides by nick translation was performed as described by BRL Life Technologies, Inc. Plasmid DNA was prepared by the alkaline lysis procedure of Birnboim & Daly (1979). Large-scale purificat’ion of plasmid DNA was performed using a Triton/sucrose lysis procedure a,nd CsCl/ethidium bromide equilibrium density gradient centrifugation (Meagher et al., 1977). Chromosomal DNA was prepared as described by Maniatis et al. (1982). except that Tris-EDTA buffer at pH 9 inst,ra,tl of pH 8 was used for the step of extraction of DNA with phenol. (c) Northern blot anulysis E. coli (Aiba et analysis Maniatis

bulk RNA was extracted with phenol at fiO”(’ al.. 1981). Northern RNA-DNA hybridization was performed essentially as described by et al. (1982). After electrophoresis through 1.3 “/,,

agarose containing transferred

6.7 “;, formaldehyde,

to a nitrocellulose

Table 1 E. coli strains Strain or plasmid

Relevant

and plasmids

genotype

or phenotype

Source or reference

JE6265 JE6266 N99 K38 MW3 MW4 MU’5 MW6 MU’7 MWlOt MWllt MW12 MW13 MW14t MW15t MW16 MW17 MU’21 YKllOO YK1220 YK1130 YK1340

recB21 recC22 sbcB15 thr thi leu his pro arg rpsL pur trp lys proC leu thi rpsL JE6265 polA11 gal HfrC@) MO287 hupBl1 (CmR) MO287 hupAl2 (KmR) argE : : TnlU MO287 hupA 16 (KmR) JE6265 pro + MW6 hupBl1 (Cms) JE6265 pro+ argE : : TnlU MWlO hupBl1 (CmR) MWlO hupA12 (KmR) MWlO hupAl2 (KmR) hupB1 I (CmR) JE6265 pro+ argE : : TnlO MU’14 hupBl1 (CmR) MW14 hupA16 (KmR) MW14 hupA16 (KmR) hupBl1 (CmR) JE6266 hupBl1 (CmR) w3110 trpc9941 YKllOO hupBl1 (Cms) YKllOO hupA16 (KmR) YKllOO hupAl6 (KmR) hupBl1 (CmR)

A. Nishimura A. Nishimura M. Gottesman K. Horiuchi This work This work This work This work This work This work This work This work This work This work This work This work This work This work (i. Yanofsky This work This work This work

Plasmid pACYCl77 pACYC184 pMW4 pMW7 pMW8 pMWl1 pMWl2 pTK20

KmR ApR P15A replieon Cms TcR P15A replicon ApR hupB+ pSR322 replicon ApR hupB+ pUC9 replicon pMW7 hupBl1 ApR TcR hupA + pSR322 replicon pMWl1 hupAl2 ApR hupA+ pBR322 replicon

Chang & (‘ohen (1978) Chanp & Cohen (1978) Kano et al. (1986) This work This work This work This work Kano ef al. (unpublished)

MO287

Winans et al. (1985)

RNA

was

filter (Schleicher & Schuell,

= JC7623

t The genotypes of MWlO and MWll are the same as t)hose of MW14 and MW15, respectively. However, the two sets of strains were isolated independently.

hupA-hupB

Deletion

Mutants

Inc.), and the immobilized RNA was hybridized at 42°C in buffer containing 50% formamide to 32P-labeled oligonucleotide probes specific for the hupB or hupA gene. The region probed was nucleotides 33 to 59 from the 5’ terminus of the coding region (Kano et al., 1985, 1987) in both genes. (d) Preparation

and electrophoresis of protein

Whole-cell lysates were prepared as described by Wada et al. (1986). HU protein was partially purified frotn this lysate as follows. The whole-cell lysate was washed with 100% acetone, and the dried precipitate was resuspended in buffer A (0.02 iv%-Tris. HCl (pH 81), O-05 M-NaCl, 5 mM-Na,EDTA, 1 mhr-/I-mercaptoethanol). The protein suspension was heated at 100°C for 10 min. After removing the insoluble material by centrifugation, the supernatant was applied to a column of cellulose containing calf thymus DNA (Sigma) as ligand. Proteins were eluted with 2 M-NaCl in buffer A, precipitated with 10% trichloroacetic acid, and washed with acetone.

Chromatography on pl 1 phosphocellulose (Whatmann) was performed in buffer containing 10 mm-Tris. HCl (pH 7.5). 1 mM-Na,EDTA and 60 mM-NaCl. The fraction containing HU protein was eluted with 2 M-NaCl, precipita.ted with 10% trichloroacetic acid, washed with 100% acetone, and resuspended in buffer containing 25 y0 acetic acid, 2.5 M-urea, 1% Triton X-100 and 0.01 y’ pvronine Y. Protein samples were analyzed by Triton/ acid/urea/polyacrylamide gel electrophoresis (Zweidler, 1978; Rouviere-Yaniv & Kjeldgaard, 1979). After electrophoresis, proteins were visualized by Amido Black staining.

of Escherichia

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3. Results (a) Construction mutant

of cloned, hup genes

Our strategy for constructing bacterial strains carrying mutations in the hup genes was first to construct the desired mutations on cloned copies of the genes and then to replace the wild-type chromosomal hup genes with the mutant alleles. For this purpose, we made mutant hup alleles by replacing part or all of the coding region of the cloned genes with genes encoding selectable drug resistance. To construct the mutant hupB allele, a 2.9 kbt BarnHI-PstI DNA fragment containing the hupB gene was cloned into pUC9, generating plasmid pMW7. There are two unique restriction sites in or near the hupB gene on plasmid pMW7: an EcoRV site 45 base-pairs downstream from the initiation codon and an AatI site 125 base-pairs downstream from the stop codon of the gene. The EcoRV-AatI fragment (0.4 kb) of pMW7 was deleted, and a 1.4 kb Hue11 DNA fragment containing the chloramphenicol-resistance (CmR) gene of pACYC184 was inserted into the cleaved site using BgZII linkers (generating plasmid pMW8). The mutant hupB allele thus constructed (Fig. l(a)) was designated hupBl1. t Abbreviations base-pair(s).

used: kb. lo3 bases or base-pairs; bp,

( b)

Figure 1. Structures of the hup gene mutations. Open boxes represent the coding regions of the hup genes; filled boxes represent genes encoding CmR (chloramphenicol resistance) and KmR (kanamycin resistance); hatched boxes represent the flanking regions of the drug-resistance gene; continuous lines represent E. co& chromosomal DNA; and filled bars represent the regions corresponding to the probes used for Southern hybridization. (a) The hupBl1 mutation; (b) the hupA mutations. Relevant restriction sites are indicated, and distances are given in kb.

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M. Wada et al.

To construct mutant hupA alleles, a 12.5 kb EcoRI fragment containing the hupA gene was cloned into pRR322 to generate plasmid pMWl1. On pMWl1, there is a unique cleavage site for KpnI in the middle of the coding region. pMWl1 DNA was linearized with KpnI, and treated with bacteriophage T4 DNA polymerase to create blunt ends. A I.3 kb Hue11 DNA fragment containing the kanamycin-resistance (KmR) gene of pACYCl77 was then inserted using BgZII linkers to create plasmid pMWl2. The mutant hupA allele thus obtained (Fig. l(b)) h as a 4 bp deletion of the HU-2 coding region and a I.3 kb insertion of foreign DNA plus the BgEII linkers (10 bp at each end). This hupA allele was designated hupAl2. The hupAl6 allele, in which hupA gene is almost completely deleted, was constructed similarly, but in this case, the KpnI-linearized pMWl1 DNA was partially digested with Ba131 nuclease before inserting the fragment encoding KmR. The BaZ31-generated deletion extends from 39 bp upstream to 42 bp downstream from the hupA coding region. The includes deleted region the transcriptional terminator but not the promoter of the hupA gene (Kano et al., 1987). of the chromosomal hup+ loci with the mutant hup alleles

(b) Replacement

We transferred each mutant allele of the hup genes into its correct location on the bacterial chromosome by transforming a recBC sbcB mutant strain with a linear restriction fragment containing the mutant allele and selecting for transformants exhibiting the appropriate drug resistance. In this technique (Winans et al., 1985), replacement of the chromosomal gene occurs via recombination between bacterial DNA flanking the selectable marker on the fragment and the homologous sequences on the chromosome. A 3.9 kb fragment containing the hupB mutation was produced by digesting plasmid pMW8 with BamHI and Hind111 (Fig. l(a); the Hind111 site is 12 bp from the PstI site downstream from hupB). Transformation of st,rain MO287 (recB21 recC22 sbcBl5) with the digested pMW8 DNA produced CmR transformants at a frequency of about 130 transformants/pg linear DNA. Replacement of the chromosomal hupA + locus with the hupA12 mutation was carried out by transforming strain MO287 with EcoRl-digested pMW12 DNA. EcoRl cleaves the plasmid 4.4 kb upstream and 6.9 kb downstream from the coding region of the disrupted hupA gene (Fig. l(b)) and excised the entire fragment of bacterial DNA used for cloning hupA. KmR transformants were isolated at a frequency of about 230 transformants/pg linear DNA. Substitution of the chromosomal hupA + locus with the hupAl6 allele in a similar manner produced KmR transformants at a frequency of about 110 transformants/pg linear DNA. Construction of hupA-hupB double mutants was carried out by transducing the hupA12 or hupAl6

mutation into strains MWI and YKl220, which the hupBl1 mutation. Transductants carry exhibiting both KmR and CmR were obtained at, relatively high frequencies (10 to 28’j/;, cotransduction with argE genes), (see Table 3). (e) Genetic characterization of hup mutants by PI transduction

Our isolation of CmR, KmR and CmRKmR st’rains in the constructions described above indicates that loss of either one or both of the hup genes is not lethal to the cell. To support this conclusion. we confirmed that the mutant’ hup genes had actually replaced the hup+ genes on the chromosome and eliminated the possibility that t,he mutant. genes had been integrated elsewhere, leaving the original hupB+ or h,upA + allele intact. We firsts analyzed the chromosomal locations of the buy alleles by determining their frequencies of caotransduction with markers known to be closely linked to the hup loci in wild-type strains. The h.up mutations were transduced from their respective MO287 derivatives into the strains listed in Tables 2 and 3, and the frequencies of cotransduction with pro(’ in t)he cast of hupB1 I, and with argE : : TnlO in t,he case of the hupA mutations, were determined. Table 2 shows that the CmR phenotype of hupBl1 was cotransduced with pro0 at relatively high frequencies compared wit,h t,hose obtained bet’ween proC and hupB+ (Kano et a,l.. 1986). supporting t,he idea that the hupBl1 mutation is integrated int,o the native hupB locus. Similarly, Table 3 (A and H) shows that the KmR phenotype of the hupA 12 and hupA 16 efficiently with wa4: cotransduced alleles argE : : TnlO (TcR), supporting the idea that the chromosomal hupA+ gene was, in fact, replaced by those mutations. The hupBl1 mutation was transduced at comparable frequencies into strain JE6265 and into JE6265 transformed with plasmid pMW4 (Table 2). The presence of pMW4 results in about a ninefold higher level of HI;-1 protein than normal (unpublished result). Essentially similar results were obtained for transduction of the hupA mutations into strains MW7 or MWl I with and without plasmid pMW4 (Table 3). Therefore, the HI: protein it~self did not significantly affect transduction of these marker genes. Relatively high Table 2 Linkage

Kecipient

analysis

by PI phqe (‘otransduction

(‘mRpY)+( 0”) JE6265 JE6265

[pMW4]

I l/R10 (i/240

(3-5) (2.5)

transduction of’ ( ‘mR and Pro++

Pro+i(W( 9”) -24jlOO 7/M)

(24)

(14)

Transduction analysis was carried out using the method Miller (1972). Strain MW3 was used as the donor. t Pro+ or CmRtransductantswereselected,and the number (!mR or Pro+ c&mies. respect,ively. were scored.

of of

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Deletion

Mutants

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analysis

A. &transduction

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frequencies than normal (Kano et al., 1986). Similarly, the transduction frequencies obtained for the mutant hupA alleles (Table 3) suggests that no in these has arisen mutation suppressor transductants either.

Table 3 Linkage

of Escherichia

of Km* and Tc*t

Recipient Km*/Tc* MWB MW7 MW7 [pMW4]

(%)

(d) Southern blot hybridization analysis of hup mutant genes

18/120 (15) 19jl20 (16) 12/120 (10)

The location of the mutant hup genes in the bacterial genome was examined in greater detail by Southern hybridization analysis. An EcoRV-AatI DNA fragment (0.4 kb) covering the coding region and an AccII-AccII DNA fragment (144-base) covering the Pl promoter region of hupB (Fig. l(a)) were used to probe chromosomal DNA isolated from a hupB strain and from its hupBl1 (CmR) derivative. The results presented in Figure 2(a) show that the CmR strain did not contain DNA homologous to the 0.4 kb EcoRV-AatI probe (compare lanes 2, 4 and 6 with lanes 1, 3 and 5) but did contain DNA homologous to the 144-base probe (see lanes 8, 10 and 12). Moreover, the fragments that hybridized to the 144 base probe exhibited the sizes expected for the hupBl1 allele with its 1.4 kb insert encoding CmR (Fig. l(a)). These results confirm that the hupB11 mutation has replaced the wild-type hupB gene on the chromosome. The O-4 kb EcoRV-AatI probe hybridized to two fragments in the HpaI-CZuI digest of DNA from strain (Fig. 2(a), lane 5). The larger the hupB+

B. Cotransduction

of Tc* and Krnst

Recipient Tcs/Km* MWlO MWll MU’1 1 [pMW4]

(%) (18)

7/40 s/40

(13)

llj40

(28)

Transduction analysis was carried out using the method of Miller (1972). Strains MW4 and MW5 were used as the donors for cross A and 1%.respectively. tTc* (A) or Km* (B) transductants were selected and the number of Km* (A) or TcS (B) colonies were scored.

of CmR pro+ frequencies of appearance transductants (Table 2) suggest that viability of the hupBl1 transductants is not due to the presence of If viability required some suppressor mutation. acquisition of a suppressor mutation, we would expect to obtain CmR transductants at much lower

(0)

(b) I

2

3

4

5

6

7

8

9

IO

II

12

4.6 2.6

HUPS

2. I

5.8 5.5 4.5

I.6 1.4

Figure 2. Southern blot hybridization analysis of hupB and hupA mutants. Chromosomal DPU’A from strains JE6266 (wild-type) (lanes 1, 3. 5, 7, 9, 11 in (a)), MW21 (a JE6266 derivative carrying the hupBl1 mutation) (lanes 2, 4, 6, 8, 10, 12 in (a)). MW14 (wild-type) (lane 1, 5 in (b)), MW12 (hupAl2) (lanes 2, 6 in (b)), MW16 (hupA16) (lanes 3, 7 in (b)) and MW17 (hupB11 hupA16) (lanes 4, 8 in (b)) were digested with BamHI and CZaI (lanes 1, 2, 7, 8 in (a)). EcoRI and ClaI (lanes 3. 4. 9, 10 in (a)), HpaI and CluI (lanes5, 6, 11, 12 in (a)), ClaI (lanes 1, 2, 3, 4 in (b)), and BamHI and PstI (lanes 5, 6, 7, 8 in (b)). After electrophoresis, the digested DNAs were hybridized with 32P-labeled probes: an EcoRVAatI fragment covering most of the hupB coding region (see Fig. l(a). lanes 1 to 6), an AccII fragment homologous to the hupB promoter (see Fig. l(a), lanes 7 to 12) or a CZaI fragment covering the hupA coding region (see Fig. l(b), lanes 1 to 8). The molecular sizes corresponding to each DNA band are indicated. The bands labeled HupA in (a) correspond to a CZaT fragment containing the entire hupA coding region (see Fig. l(b)), and the bands labeled HupB in (b) correspond t,o the CZaI (lanes 1 to 4) and the P&I (lanes 5 to 8) fragments containing the hupB gene (see Fig. l(a)).

M. Wada et al.

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band corresponds to the 1.2 kb ClaI fragment of hupA (Fig. 1 (b)). Our hybridization conditions apparently permitted detection of the partial homology (7076,; Kano et al.. 1987) between the hupB and hupA region. hw,pA (lonfirmation that the KmR-encoding, mutant alleles had indeed replaced the wild-type hupA gene on the chromosome was also obtained by DNA-DNA hybridization (Fig. Z(b)). We used a 1.2 kb CZaI DNA fragment that covers the hupA coding region (Fig. l(b)) to probe CZaI digests of chromosomal DNA isolated from a hupA + strain and from its KmR (hupA12 or hupAl6) derivatives. Hybridization to two distinct’ bands was observed in the DNA from the KmR (lanes 2 to 4), whereas only one band was detected in the DNA from the hupA+ strain (lane l), indicating that the hupAl2 and hupA16 alleles had been integrat’ed into the chromosomal locus by dividing and replacing the coding region. The CEaI fragments detected in DNA from the strains carrying t,he complete hupA deletion, hupA16 (lanes 3 and 4), were smaller than the fragments detected in DNA from the hupAl2 mutant (lane 2) by about the amount expected. Only one fragment homologous to the probe was detected in each strain when BarnHI-PstI digest’s of DNA from the hupA + (1an? 5) and hw,pA mutant, (lanes 6 to 8) strains were analysed. The fragments detected in DNA from the hupAl6 mutants (lanes 7 and 8) were slightly but discernibly smaller than the fragment detected in DNA from the hupi212 mutant (lane 6).

(e) The hup mutants are de&Sent it1

the production

D D

proteins

Southern blot hybridization analysis of b’:. co/i DNA with the hupB and hupA J)X;A probes indicabes that there is only one gene for FLU@ and one gene for hupA on the chromosome (dat,a not, shown). Therefore, our h,up deletion mutants should be deficient. in the production of HI’ proteins. This possibility was examined by analysiny the tnKNA and protein products of the hup genes in thch mutants. of RX A samples After gel eleatrophoresis prepared from a wild-t,ype st,rain and from its hupBl1, hupA 16 and hupBI 1hupA 16 derivatives. the RNAs were hybridized with a 27-mer synthetic oligonucleotide whose sequence is homologous to part) of the coding regions of both the hupB and hupA genes. Figure 3 shows t,ha.t hupH mRNA is not detected in strains carrying the hupB1 I mutation (lanes2 and 4) and that hupA mRNA is not found in strains carrying the hupAl6 mutation (lanes 7 and 8). The hupB gene possesses two t,andem promoters whose efficiencies in initiating transcription are comparable (unpublished results). resulting in production of two molecular species of mRNA (Fig. 3, lanes 1 and 3). On the other hand. t,he h,upA gene is transcribed from a single promoter (Fig. 3, lanes 5 and 6; and see Kano Pt al.. 1987). The production of HI’-1 and HIT-2 proteins in the hup mutants was also examined. After partial purificatjion. H 1: proteins from parental and

5

flupB mRNA

qf HI!

6

7

8

bupA mRNA

Figure 3. Northern blot hytnidization analysis of the ?~uptranscripts in kupB and hupd deletion mutants. Tot,ai RNA from strains MW14 (wild-type) (lanes 1, 5), MW15 (hupB11) (lanes 2, 6), MWl6 (hupA 16) (lanes 3, 7) and LMW17 (hupR11 hupAl6) (lanes 4, 8) was hybridized with 32P-labeled. 27-mer oligonucleotide probes corresponding to the 33rd to the 60th nurleotide of the hqH (lanes 1. 2, 3, 4) or the hupA (lanes 5. 6. 7, 8) roding regions (Kano it nl.. 1985. 1987). The hupA and hupH coding stquences are highly homologous except for this region (Kant) pf (~1.. 1!)85. 1987).

I

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Deletion

3

4

Mutants

of Escherichia 6

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coli 7

6

I I 6

/ (

HU-I

E

Figure 4. Electrophoretic analysis of HU proteins synthesized in hup mutants. HU protein was partially purified from strains YKI 100 (wild-type) (lane l), YK1220 (hupB11) (lane 2), YK1130 (hupA16) (lane 3), YK1340 (hupRl1 hupA16) (lane 4). YKllOO harboring pMW4 (HU-1 overproducer) (lane 5) YKllOO harbouring pYK20 (HU-2 overproducer) (lane 6) and YK 1100 harbouring pBR322 (lane 7), and electrophoresed as described in Materials and Methods. Proteins were visualized by Amido Black staining. Purified HU proteins are in lane 8. The HU-1 and HU-2 protein bands are indicated by arrowheads. mutant strains were analysed by Triton/acid/urea/ polyacrylamide gel electrophoresis (Fig. 4). The HU-1 and HU-2 protein bands were identified by co-migration with the completely purified HU proteins from a wild-type strain (Fig. 4, lane 8). As expected, the hupA mutants did not produce detectable amounts of HU-2, and the hupB mutant did not produce detectable amounts of HU-1 (Fig. 4, compare lanes 2 to 4 with lane 1). Upon introduction of an HU-overproducing plasmid carrying either the hupB or the hupA gene into the wild-type strain, a marked increase in the amount of HU.-1 (Fig. 4, lane 5) or HU-2 (Fig. 4, lane 6), respectively, was observed. Deletion of the hupB gene resulted in an increased level of HU-2 protein. In addition, the level of HU-2 protein decreased upon overproduction of the HU-1 protein in a strain carrying the hupB plasmid, pMW4. These observations and others suggest that HU protein may regulate the expression of the hupA gene (unpublished results). The levels of the HU proteins in total cell lysates prepared from parental and hup mutant strains were also examined by Western blot analysis using anti-HU antibodies. The results obtained were in agreement with those presented above (data not shown).

2,o

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0.05

o-01

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(f) Physiological

hupA-hupB

characterization of the double mutants

Success in isolating hupA-hupB double mutants suggests that both hup genes are dispensable for cell

( h )

Figure 5. Effect of hup mutations on the growth rate. Strains MWlO (wild-type, 0). MWll (hupB11, a), MW12 (hqA12, a) and MW13 (hu~A12 hupB11, A) at 37°C’. Cell growth was were grown in L-broth monitored by measuring .4,,,.

M. Wada et al.

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(a)

Figure 6. Cell morphology and nucleoid structure of’ the hupA hupH mut’ant, and its Ijarental strain. Samples wt~~ taken from cultures growing exponentially at 37°C in L-broth and visualized by fluoresrencar microscopy. (a) MWI? (wild-type);

(h) MW17 (hupA hupR).

growth at 37°C. A slight but discernible difference in growth at, 37°C between the double mutant and t,he wild-type strain is that, the mutant exhibits a I.&hour lag after dilution into fresh L-broth (Fig. 5). The rat’e of exponential growt’h (measured as absorbance at 550 nm) of the double mutant at) 37°C was comparable to the growth of the wildtype and buy single-mutant st,rains. Viable cell number per ,4,,, unit of the double mutant was. however. reduced to one-fifth of the normal value under t’hese growth conditions. All hup double mutants examined so far. in four different backgrounds (JE6265, W3110, N99 and HfrC(I)). exhibited essentially similar growth characteristics. When morphology of the cells was investigated by visible-light microscopy, we found t,hat the cells carrying the hup double mutat,ion were filamentous. Such a morphology was not observed in hup single mutants. We then examined the distribution of the nucltoid in wild-type and h?yA 16.J~upRll mut,ant. cells. The cells were collected from cultures growing exponentially at 37°C in L-broth and observed by fluorescence microscopy after staining of the DNA with DAPT. Masses of DNA of unequal sixes wet-cl irregularly distributed in the filamentous cells

obtained from the culture of the /~u+,;l -/u~‘N doublt~ mutant (Fig. 6(b)). 111 addition. ;t c~onsidcrablr number of DNA-less (*ells from this culture ~VI’P observed in the microscope as masses cmitt,ing kissintense fluorescence. These anucleate ~~~11swere nearly normal in size and represented roughly one third to two-thirds of the cell populat,ion. Thrscobservations indicate that absenccx of’ t.hcL HI’ protein causes structural abnormalitv of’ t hr nucleoid and, by some unknown mec?hanisrn. kads to the disturbance of chromosome part)itionirlg and cell division. Consistent with these observations. the viable cell number and DNA content (nortna~lized to absorbance at 650 nm) of the huprl -hupN mutant (MW17) were 15 t)o ‘LOY;, and 30 10 60’),,. (MWl4) IPV&. respectively, of t,hr wild-type depending on the stage of caell growth. We also found that the hupA -//,upH clouhlt~ exhibited a (2old-sensitive growth mutant phenotype. As shown in Table 4. tht, tloubling t irntb of a h,up,4 -hupt3 mut,ant at tfmperatJures lower t hatl 26°C’ was cbonsiderably longer than t,hat, of t hr will1 type st,rain at the same temperature. Sucah stressful conditions as colti-shock wertx observchd ttr af%ct CY~II physiology significantly. When viable- (~11 nurr~b~

hupA-hupB

Growth

rates

of

Table 4 hup mutants at various

Deletion Mutants of Escherichia

.

hupAB

30

60 27 31 60

26

55

23

70

60 27 31 55 83 125

46

42 I31

.-.-.-.-.0. IT--l.

hupAB/hup+f

\-.I-

-.

I-’

*.-

\

1.0 1.0 1.0 1.1

hup+, 46 -*z. .C. --2-u hupAE, 46

-2.

.

*.

Doubling time (min)t hup +

589 Heot shock

temperatures I

Temperature ( “C)

coli

-

OS hup+, 48

10-l

1.5

1.8

tCells of YKllOO (hup+) and YK1340 (hupAB) were grown in at the temperatures indicated. Doubling time was measured by monit)oring the optical density of the cell cultures at 550 nm. $ The ratio of doubling times is given. Lhroth

was measured after treatment of hupA-hupB mutant cells at 0°C for short periods (1 to 4 h), 70 to 90%) of the cells were found to have been killed (Fig. 7). The double mutant is also sensitive to heat shock. Treatment of hupA-hupB mutant cells at 48°C for 30 minutes reduced the viable cell number t,o a level low3 that of the control (Fig. 8). It appears, therefore, that the hupB and hupA genes are dispensable for cell growth at 37”C, but essential for growth under some conditions of stress.

0 2 : 2 lo-’

I o+

1 0

k

1

I

I

I

c

5

IO

15

20

25

30

Incubation

(min)

Figure 8. Effect, of heat-shock on survival of the hupA 16-hu$l l double mutant. The conditions and symbols used are the same as described in the legend to Fig. 7, except that the cells were incubated at the temperatures indicated (in “C), instead of at 0°C.

Cold shock

4. Discussion

\

o-o hfJp+

0

hupA-hupB

\ O-0-a

0

I

2 lncubotion

3 at O’C

4 (h)

Figure 7. Effect of cold-shock on survival of the hupA16-hupB11 double mutant. Strains YKllOO (wildtype. 0) and YK1340 (hupA16-hupB11, 0) were grown t>o log phase in Lbroth, shifted to 0°C for various times, and plated on L-broth agar. The plates were incubated at 37°C for 2 days. and the number of colonies was scored. (Colony number immediately before the shift, has been taken t)o equal 1.0.

HU protein is thought to play many important roles in the cell (Drlica & Rouviere-Yaniv, 1987). To clarify whether HU protein is required for bacterial cell growth and to investigate the function(s) of the protein in viva, we constructed chromosomal deletion mutants of t’he hup genes. Each chromosomal hup+ gene could be replaced easily by homologous recombination with its deletion derivative that had been constructed on a plasmid in vitro. Moreover, the hupA chromosomal deletions could be transduced easily into a strain carrying the hupB chromosomal deletion, strongly suggesting that both the hupB and h.upA genes are non-essential. Southern blot analysis of chromosomal hup DNA, Northern blot analysis of hup mRNA and polyacrylamide gel analysis of cell extracts all confirmed that the hupB (CmR) hupA (KmR) double mutants constructed bv PI -mediated transduction are actually HU-deficrent. Thus we concluded that neither HU-1 nor HI!-2 protein is essential for cell viability. The possibility that the expression of some gene(s) other than hup compensates for the deficiency in hup gene activity cannot be ruled out, however. DNA binding protein THF consists of two subunits c1 and fi of about 11,000 M,, which are highly homologous with HU (32 to 37 O/Oshared amino acid sequences) (Mende et al., 1978: Laine et al., 1980; Flamm 8r Weisberg, 1985; Mechulam et al., 1985). HU stimulates

590

M. Wada et al.

recombination at Latt site when at@ has a mutation in the H’ binding site for THF. HU also stimulates TnlO transposition under the absence of IHF (see review by Drlica & Rouviere-Taniv, 1987). In addition, IHF can replace HC protein for open complex formation at oriC, and also substitutes for HU protein in the stimulation of the reconstituted replication of oriC templates (Hramhill & Kornberg, 1988). In these respects, THF may be a candidate protein for compensation for the deficiency in hup gene activity. Quantitative and qualitative analysis of proteins in the hupA-hupR double mutant might shed some light) on this possibility. The hupB or hupA single mutant did not exhibit) any phenotypes different from t,hose of the parent.. Gel filtration of cell extracts from hup single mutants showed that HIT can form a homodimer (unpublished results). This observation suggests that a homodimer of HIY may be active in viva in hup single mutants. This possibilit’y is consistent wit,h reports that. many bacterial HU-like proteins exist in a homodimer form i,n ,uivo (Drlica & Rouviere-Yaniv, 1987). The hupA -hupB double mutants, however, do exhibit mutant, phenotypes, such as filamentation (Fig. 6) and cold sensitivit’y. Filamentation was observed more frequently at temperatures below 32°C and was generally accompanied by production of anucleated cells. With the strain of JE6265 used as the genet’ic background, filamentation could be observed even at 37°C (Fig. 6(b)). Filamentation of cells ha,s been known to occur if elongation of DNA is blocked by ultraviolet irradiat.ion. for example. and SOS functions are induced (Witkin. 1976). However, we think that SOS function has not been induced in the filamentous cells of the hup double mutant because filamentation does not induce a Llysogen of t,he double mut,ant (unpublished results). The product.ion of filamentous and anucleate cells by the h,up double mutant appears. instead, to be similar to the phenotypes shown b? rlna(ts) mutant,s (Hirota et al.. 1968: ,Jaffe et al.. 1986: Xorris et al., 1986; Orr ef nl., 1979) when I)KA replication is inhibited. Growth of hupA-h,upll double mutant cells is cold-sensit.ivr at t,emperatures below 30°C and is also sensitive to cold shock at 0°C‘ or to heat shock at, t,emperatures higher than 48°C. These phenomena suggest that HI! protein plays an important role in protect,ing nucleoid structure from physiological damage caused hy temperature-related st.ress, although HU protein IS not a member of t,he heat-shock proteins. Tt has been report,ed that heating E. coli to 50°C resulted in measurable changes in nucleoid structure, resulting in an increased sedimentation coefficient and a broadening of the sedimentation profile. When the cells are transferred to 37°C. the nucleoid structure is repa,ired before cells can resume normal growth (Pellon pt a/., 1982). From this point of view. it is interesting that HU protein is a major protein component of bhe nucleoid, which constitutes about 25Yb weight of t,otal nucleoid proteins (Varshavsky

1977); and maintains higher-order structure of DNA (Hroyles ct Pettijohn, 1986). Cold-sensitive mutations have heen isolated in several genes of E. c.oli. Some of these mutations result in deficiencies in the formation of such macromolecular complexes as ribosomes (Jaskunas et a,l., 1974). t’he DNA replication init)iation complex (Hansen et nl., 1984). and the translation initiation complex (Shiba rt 1~1.. 1986). Formation of macromolecular caomplaxes has been thought, to require higher temperature in thr mutants than in wild-type strains. Similarly, HI!deficient cells might require temperatures higher than normal to assemble and/or maintain nucleoid stru&urr. It has been reported t,hat, replication of DXA from B. di oTj(’ is dependent, on the HI’ prot,ein it, Gtro (I)ixon CyrKornberg, 1984: Ogawa rt al., 1985). Our ohservation that the DNA content in CGM 01 the hup double mutant was reduced rrlat,ive to t’hat. of the hup+ strain and that, the majority of cells artA filamentolls or anucleate may reflect. a requiremcnl for HTT in DNA replication and/or DXA partitioll. The possibility that HU is actually involved in thp initiation of replication i,n LGM~has been supported by the finding that mini-F pla,smids c.annot double mutant under replicate in an hupA-hupH conditions in which repli: is expressed (unpublished results). Our additional finding that, the broad host range plasmid RSFlOlO is not’ able to transform thr hzLy double rnut’ant (unpublished rrsu1t.s) suggests that HIT-likt proteins (Drlica & Rouviere-Yani\-. 1987) may be involved in DNA rcplicat ion of su(+ plasmids. (‘raigicb fjt (11. (1985) have reporl~~tl that. f I’~I~sposit ion of Mu phage DNA in /,itro rtsquires H 1’ prot,tGn. i\‘e havta found t.hat. Mu phage requires either HI’- 1 or H I’-2 prot’cin for replicatircx transposition in E. coli in IGO (unpublished rrsult s). Johnson et r*l. (1986) have reported that inversion of’ SaEmorc& H region l)T\‘A in E. roli-based cell-frt~ systrm requirts E. coli HlT protein. Our studies of I>h’A inversion i?j r*ic>o havra rttvealed t#hat H 1.. prot,ein apparently stimulates the inversion of the (: region of Mu phage DSA and the P region of F:, co/i e14 I)lXA in addition to t,he H region of Srtlnwdlo (unpublished results). Insertion and deletion mutants of huyN yens’ have been rrportrtl reeent’ly (Storts R- Markoyitz. 1988). No rffect, however. was observed on t.hrac*rll physiology. including cell growth, phage replicaat ion and phage lysogenization. Thrsr observations arcA c>onsistrnt. wit,h our result>s showing that, t,he cella in which either the //zL@ or the hupl-l gerw is def&tivc grow normally. but cells in which both of the !/U/I genes arc defec*tivr exhibit phenotypes difft~rrnt from t,hc wild-type strain. Our st)udies so far have provided evidence that HI: prot,ein funct.ions in ciao in DXA replication. DNA transposition and DNA recornbinstion Studies are in progress t.0 clarify the molecular mecahanisms of those functions of HI’. et al.,

hupA-hupB

Deletion Mutants of Escherichia

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Edited by K. Matsubara