Expression of F transfer functions depends on the <i>Escherichia coli</i> integration host factor

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Article

Reference

Expression of F transfer functions depends on the Escherichia coli integration host factor

GAMAS, P., et al.

Abstract

We present evidence that the Escherichia coli DNA binding protein, IHF, plays an important role in conjugal transfer of the plasmid F. Our results suggest that IHF exerts this effect by positively effecting transcription of the transfer (tra) operon of the plasmid.

GAMAS, P., et al . Expression of F transfer functions depends on the Escherichia coli

integration host factor. Molecular and General Genetics , 1987, vol. 207, no. 2-3, p. 302-305

DOI : 10.1007/BF00331593

Available at:

http://archive-ouverte.unige.ch/unige:149377

Disclaimer: layout of this document may differ from the published version.

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Expression of F transfer functions depends on the Escherichia coil integration host factor

P. Gamas 1, L.

Caro 2,

D. Galas 3, and M. Chandler s

1 Centre de Recherche en Biochimie et Genetique Cellulaire du C.N.R.S., 118 Rte. de Narbonne, F-31062 Toulouse Cedex, France 2 Departement de Biologic Moleculaire, Universit6 de Geneve, 30 Quai Ernest Ansermet, Geneva, Switzerland

3 Department of Molecular Biology, University of Southern California, Los Angeles, CA 90089-1481, USA

Summary. We present evidence that the Escherichia coli DNA binding protein, IHF, plays an important role in con- jugal transfer of the plasmid F. Our results suggest that IHF exerts this effect by positively effecting transcription of the transfer (tra) operon of the plasmid.

Key words: Escherichia coli- IHF protein- Plasmid F - Conjugal transfer

Although not essential for cell viability, the heterodimeric Escherichia coli protein, IHF, a bacterial type II DNA bind- ing protein (Flamm and Weisberg 1985; Mechulam et al.

1985; Miller 1985), is implicated in a variety of biological processes. These include: site specific recombination of lambdoid phages (see Bushman et al. 1986); modulation of gene expression both at the transcriptional (Friden et al.

1984; Krause and Higgins 1986) and translational (Ma- hajna et al. 1986) levels, and establishment and/or mainte- nance of the plasmid pSC101 (Gamas et al. 1986).

We report here that IHF has a strong effect on another process in E. eoli: conjugal transfer of the plasmid F. We provide evidence that IHF stimulates, directly or indirectly, the expression of certain plasmid-encoded genes necessary for transfer. In particular, IHF affects both the synthesis and/or assembly of sex pili and the expression of genes involved in surface exclusion. Since both sets of genes form part of the same operon, the tra operon (for review, see Willetts and Skurray 1980), it seems probable that IHF is required for expression at the transcriptional level.

A series of experiments in which the frequency of transfer of the F-derived plasmid pOX38Km (a plasmid composed of the largest HindIII fragment of F together with a HindIII fragment encoding resistance to kanamycin;

Guyer et al. 1981) was measured from a set of strains (Ta- ble 1) isogenic except for the presence or absence of muta- tions in one or both of the IHF genes, himA and hip.

Transfer from the wild-type strain MC240 is approximately 500-fold higher than that from either the himA (MC251), hip (MC252), or himA/hip double mutant (MC253) (Ta- ble 2).

To confirm this result, we introduced both mutations, by P1 transduction, into a second genetic background Offprint requests to: M. Chandler

(C600). The difference in mating efficiencies of pOX38Km from C600 and its I H F - derivatives was similar to that observed for MC240 and its I H F - derivatives (compare C600 and MC422, for example).

To assess whether the phenomenon is restricted to pOX38Km, we determined the behaviour of an intact F plasmid marked with the kanamycin transposon Tn903 (F- kan; Heffron et al. 1978). The results of such experiments are also presented in Table 2. Comparison of MC240/F-kan (wild-type) with MC253/F-kan (himA hip-) indicates a difference in transfer efficiency of over three orders of mag- nitude.

To determine whether IHF is required simply for stable maintenance of F, donor cells grown under the conditions used for mating were screened for the presence of the pOX38Km plasmid. No significant loss of the Km + gene was observed (data not shown). Indeed, prolonged growth in non-selective medium over a period of about 20 genera- tions in the presence of 0.01% SDS (to avoid retransfer of pOX38Km to cells having lost the plasmid; Helmuth and Achtman 1975) did not result in significant plasmid loss (data not shown).

It has been shown previously that derivatives of F, gen- erated by fusion in vitro (Johnson and Willetts 1980) or in vivo (Galas and Chandler 1982) with the high copy number plasmid pBR322, have an increased copy number and show an increase in transfer efficiency compared with the F plasmid alone. We analysed the effect of IHF on the transfer properties of such high copy number cointe- grates. We used two types of plasmid: pBXI, constructed in vitro by insertion of HindIII-cleaved pBR322 into the unique HindIII site of pOX38, and a chimaeric plasmid, pOX38Km: : pOC 15, constructed in vivo by IS 1-mediated replicon fusion between pOX38Km and a pBR322 deriva- tive plasmid, pOC15 (Gamas et al. 1985). Transfer data presented in Table 2 show that, in both cases, the reduction in transfer efficiency caused by the absence of IHF is com- pensated by an increase in F copy number (compare MC240/pBX1 with MC253/pBX1 and MC294/pOX38Km- : :pOC15 with MC296/pOX38Km: : pOCl5). In the latter case the host strains carried a deletion of the recA gene to reduce homologous recombination between the directly repeated copies of IS/ at the pOX38Km-pOC15 junctions (a consequence of an ISl-mediated replicon fusion event).

Since recA- IHF- strains exhibit reduced viability, an effect

exacerbated by the presence of the cointegrate (unpublished

observations), the transfer values obtained for this series

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303 Table 1. Bacterial strains

Strain Genotype MC240

MC251 MC252 MC253 MC253A MC294 MC296 C600 MC422 LC 1006 OF4 LN 1700

ara del (lac pro) gyrA metB argE rif thi supF MC240 de182 (himA) :: TnlO

MC240 del3(hip) : cam

MC240 de182(himA) del3(hip) : cam MC253 :precise excision of TnlO MC240 del306(reeA) srl: : TnlO MC253A del306(recA) srl: : TnlO thi leu thr lacy tonA supE44

C600 de182(himA) : :Tnl0 del3(hip) : cam C600thyA gyrA r~fPl + 2 r

C600 recA rpsL

HfrH thr: :Tnl0(Sp r Sm r Tc s)

IHF and recA mutations were introduced by PI transduction se- lecting for the associated antibiotic resistance. The TnlO derivative carried by LN1700 has an in vitro insertion of a gene specifying resistance to spectinomycin and streptomycin. It was kindly pro- vided by J-M Louarn

Plasmids pOX38 pOX38Km

pBXI

pOX38Km: : pOC15 pMP3

Largest HindlII fragment of F (Guyer et al.

1981)

pOX38 carrying a HindIII fragment encoding resistance to kanamycin (Galas and Chandler 1982)

pOX38/pBR322 chimaera joined at the unique HindIII site in each plasmid (this work) An ISl-mediated fusion of pOX38Km and the pBR322-based plasmid pOC15 (Gamas et al. 1985; this work)

pBR322-based plasmid carrying a gene encoding resistance to spectinomycin and streptomycin (Prentki et al. 1987)

1000 800 600

400

200

o

1000

8oo iiii:ill N

i!:i:!i:i

°°°iii!iiill

iii:i:i:

,°°ii

°°ii

IttF + recA- p0X3gKm

~-!i:::t

i:i

123456_>7

IHF + rec/- pOX-coint.

012345827 IHF- reeh

p0X38Km

IHF reeA (NmA, ~r.D)

pOX ~ eoint.

0

:!iiii!ilili

0 I 2 3 4 5'8'->7 0 1 2 3 4 5'8'27

Fig. 1. Histogram showing the distribution of pili. The abscissa represents the number of cells carrying one, two etc. pili. Cells were grown to mid-log phase, and the pili were coated with male specific phage MS2 and prepared for electron microscopy as described (Caro and Schn6s 1966). 103 cells of each strain taken from random fields were scored for the presence and number of pill The data presented are from MC294 (IHF ÷ recA-) and MC296 (IHF- recA -) carrying pOX38Km or pOX38Km: :pOC15

of strains is probably not directly comparable with that obtained with the recA ÷ derivatives.

Since F pili constitute the receptor site of male specific phage (Caro and Schn6s 1966), we also determined the ability of the phage M13 to grow on the various strains (Ta- ble 2). The efficiency of plating of M13 on IHF cells carrying pOX38Km is greatly reduced compared to that found with isogenic IHF ÷ cells (compare MC240/pOX38Km with MC253/pOX38Km and MC294/pOX38Km with MC296/

pOX38Km). The plating efficiency on IHF- cells which carry high copy number F derivatives (MC296/pOX38Km :: pOC15 and MC253/pBX1 is, however, normal. Although no single M13 plaques could be observed in the IHF- pOX38Km-carrying strains, the cells are not entirely resistant to the phage. Using a spot test, partial lysis of the cells occurred at relatively high phage concentrations. This is in contrast to the complete resistance observed for F cells and presumably reflects the fact that some F transfer occurs from the IHF- strains. This result correlates well with the transfer efficiencies of the various strains described above and suggests that IHF is required for the synthesis or assembly of F pili. We note that growth of M13 on the IHF strains carrying the cointegrate indicates that IHF is not required for replication or packaging of the phage.

That the resistance to male specific phage is due to the absence of pili was confirmed by electron microscopy. The

results are illustrated in Fig. 1 and summarised in Table 2.

Thus pilus formation is severely impaired in the IHF pOX38Km-carrying strains MC251, MC252, MC253 and MC296 compared with the wild-type parental strains MC240 and MC294 (Fig. 1) whereas in an identical genetic background in the presence of high copy number cointegrate plasmids (e.g., MC296/pOX38Km::pOC15; Fig. 1) pili are relatively numerous.

Twelve genes are directly required for pilus formation (see Willetts and Skurray 1980). They belong to the large tra operon, which also encodes other functions such as surface exclusion (governed by two genes, traS and traT; Acht- man et al. 1980). Transcription of this operon is controlled in a positive way by the product of the traJ gene and, in F-related plasmids, negatively by the products of the finO and finP genes. F itself is naturally finO-. Since traS and traT are not involved in pilus formation and are located distal to the pilus genes, it was of interest to determine whether IHF also affects surface exclusion.

To investigate this question, homosexual crosses were performed using, as a donor, a derivative of HfrH (IHF ÷ ; LN1700), carrying a spectinomycin resistant (sp'), Strepto- mycin resistant (sm r) derivative of the transposon TnlO in- serted into the thr locus, and transferred early by this strain.

As recipients, we used the IHF ÷ or IHF- strains (MC240 or MC253) carrying pOX38Km. Parallel matings using the isogenic recipient F-strains allowed us to determine a sur-

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Table 2. Properties of IHF + and IHF- strains

Strain Plasmid IHF Transfer efficiency" M 13 b Pili c S.E. d

240 pOX38Km + 7.5 x 10 .2 S + 250

251 pOX38Km

himA

1.3 x 10 4 R - nd

252 pOX38Km

hip

1.4 x 10 .4 R - nd

253 pOX38Km

himA/hip

1.4 x 10 4 R - 36

294 pOX38Km + 6.7 x 10-1 S + nd

294 pOX38Km: :pOC15 + 2.1 S + + nd

296 pOX38Km

himA/hip

3.7 x 10 .4 R - nd

296 pOX38Km::pOC15

himA/hip

1.8 x 10 1 S + nd

240 pBXI + 3.8 x 10-1 S nd lad

253 pBX1

himA/hip

2.0 x 10-1 S nd nd

422 pOX38Km

himA/hip

4.7 x 10 4 R nd nd

C600 pOX38Km + 2.5 x 10-1 S nd nd

240 F-kan + 5.3 x 10 -1 nd nd nd

253 F-kan

himA/hip

2.1 x 10 -4 nd nd nd

" Transfer efficiency is expressed as the number of exconjugants per donor at the time of sampling. The ratio of donors to recipients was t : 3. The recipient strain used in matings with the F-kan donor strains was C600 carrying the plasmid pMP3 (to provide spectinomycin/

streptomycin as selective markers). For the C600 derivative donor strains LC1006 was used as recipient. OF4 was used as a recipient for all other matings. Matings were for 1 h at 37 ° C using previously described growth conditions (Galas and Chandler 1982). Antibiotics were used at the following concentrations where appropriate: kanamycin (km r) 20 ~g/ml; ampicillin (Ap r) 50 gg/ml; spectinomycin (Sp r) and streptomycin (Sm~), 30 and 20 gg/ml, respectively; rifampicin (rif) 60 gg/ml; nalidixic acid (Nal) 20 gg/ml). The values represent the mean of at least four independent experiments

b Five microlitres of serial dilutions of M13 (5 x 1011 p.f.u./ml) were spotted onto a lawn of the strain to be tested. S, sensitive; R, resistant

(+) presence and ( - ) absence of pili. Quantiative results are shown in Fig. 1

d Surface exclusion index. Strains were grown to mid-log phase with aeration, followed by growth for I h without aeration. Mating was continued for 40 min at 37 ° C at a ratio of 1 donor to 2 recipient cells. Selection was for transfer of Sp r and Sm r (spectinomycin, 30 gg/ml; streptomycin, 20 gg/ml) from LN1700 as an early marker. Nalidixic acid (20 gg/ml) was used as a counterselection. The surface exclusion index is expressed as the frequency of transfer of Sp~/Sm r to the F- strains MC240 and MC253 divided by the frequency of transfer to isogenic strains carrying pOX38Km

face exclusion index (Table 2). The results show a reduction of almost ten fold in surface exclusion index in the F + I H F - recipient.

We demonstrate in the results presented here that the

E. coli

protein IHF, the product of genes

himA

and

hip,

exerts a strong positive effect on conjugal transfer of the plasmid F. This phenomenon has recently been observed to occur also with the closely related plasmid R100 (J.M Louarn, personal communication). Although we do not know at present whether the effect is restricted to plasmids of the

incF

incompatibility groups, we have demonstrated in at least one other case, that of the

incP

group plasmid RPI, that transfer does not depend on intact copies of the

hirnA

and

hip

genes. No difference in frequency is observed between wild-type and mutant donor strains ( M - F Prere, unpublished results).

Strains in which the genes for either or both IHF sub- units have been deleted show a pronounced decrease, but not a total absence, of F pili. We have not, at present, determined whether this results from a reduced ability to assemble the pilus structure or simply from a reduced ability to synthesise F pili. The observation that surface exclusion is also reduced in I H F - mutants suggests however that IHF affects transcription of the entire

tra

operon rather than translation of the various genes or assembly of the pili. It should be noted that the difference in degree of the effect on transfer (greater than two orders of magnitude) and on surface exclusion (slightly less than one order of

magnitude) may simply reflect a difference in the sensitivity of each process to levels of the respective proteins involved.

A priori, it is unlikely that IHF affects expression of the negative control elements FinO and FinP since F is naturally

finO-.

Moreover, initial results using

traJ

gene fusions indicate that IHF does not have a significant effect on the expression of the positive regulator TraJ (D. Lane, personal communication). IHF might thus directly affect

tra

operon expression or may act indirectly, for example by controlling the expression of the several host genes known to be necessary for efficient F transfer:

sfrA

and

sfrB

(Beutin et al.

1981),

cpxA

and

cpxB

(McEwen and Silverman 1980). Fur- ther studies will be necessary to determine the precise molecular role of IHF in F

tra

operon expression.

Acknowledgements.

We would like to thank E. Boy de la Tour for preparing the electron micrographs, D. Rifat for excellent tech- nical assistance, and P. Prentki for happy hour. We would also like to thank D. Lane, J.-M. Louarn, and M.-F. Prere for commun- icating their unpublished results and M.-F. Prere for constructing the plasmid pBX1. This work was supported by an "Action The- matique Programmee" C.N.R.S. (MC), grant no. 3.516-0.83 from the Swiss National Science Foundation (LC), NIH grant GM19036 (DG), and an NSF/C.N.R.S. collaborative grant (MC, DG).

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Communicated by R. Devoret

Received October 24, 1986