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Mutations in HU and IHF affect bacteriophage T4 growth: HimD subunits of IHF appear to function as homodimers
Gene, 160 (1995) 131-132 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
131
GENE 08970
Mutations in H U and IHF affect bacteriophage T4 growth: HimD subunits of IHF appear to function as homodimers (Integration host factor; ihfsite; histone-like protein; one-step growth experiment)
Barbara Zablewska and J6zef Kur Department of Microbiology, Technical University of Gdahsk, 80-952 Gdahsk, Poland Received by J. Wild: 8 October 1994; Revised/Accepted: 16 February 1995; Received at publishers: 7 April 1995
SUMMARY
The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., ~, qb80, Mu and fl) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants (hupA hupB himA and hupA hupB himD) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.
The integration host factor (IHF) of Escherichia coli is a histone-like, heterodimeric protein consisting of the products of the himA gene and the hireD~hip gene. IHF participates in various processes (see, for review, Friedman, 1988). The purpose of the present work was to test (i) whether IHF and HU are needed for coliphage T4 propagation and if so (ii) whether IHF subunits are able to function as homodimers. Absence of HU (using hupA- hupB- as host) had no effect on T4 adsorption and resulted only in a slight inhibition of phage growth (including the eclipse period, as well as the average burst size) during the first cycle of development (compare Fig. 1A and D), but the second cycle of the T4 growth was completely inhibited (Fig. 1D). Correspondence to: Dr. J. Kur, Technical University of Gdafisk, G. Narutowicza Street 11/12, 80-952 Gdafisk, Poland. Tel. (48-58) 472-302; Fax (48-58) 472-694; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); A, deletion; himA, gene encoding the a subunit of IHF; hireD, gene encoding the ~t subunit of IHF; HU, major component of the proteins bound in the E. coli nucleoid; hupA, gene encoding HUa; hupB, gene encoding HUb; IHF, integration host factor; LB, Luria-Bertani (broth); wt, wild type.
SSD! 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 2 5 2 - 9
Next, the T4 phage growth experiment was carried out in the absence of IHF protein using himA himD double mutant. The curves of intracellular phage growth at the first cycle of development in the wt cells and in the himA hireD double mutant cells were similar, except that the eclipse period was about 10 min shorter in wt cells (Fig. 1A,B). In the second cycle, the T4 growth was much lower in the himA himD double mutant cells (over 10-fold reduction) as compared to the wt cells. The adsorption of the T4 phage to the wt strain of E. coli was the same as to the himA himD double mutant (results not shown). These results suggest that IHF might be needed for bacteriophage T4 growth. Since adsorption of T4 was not affected in double mutants, the inhibition of phage growth in the absence of IHF protein must occur in a post-adsorption step. Complementation analysis was carried out to confirm our suggestion. Plasmid pPLhiphimA-5 (Nash et al., 1987) was used to transform himA himD double mutant cells. The curve of intracellular phage growth in this strain (Fig. 1C) was comparable to that of T4 in a wt strain (Fig. 1A). Thus, plasmid producing IHF complemented the defect of himA himD double mutant for T4 phage growth. It has not yet been reported that bacteriophage T4 growth depends on IHF function. IHF could regulate T4
132 100000.0
0
T h e i n t r a c e l l u l a r g r o w t h of the T 4 p h a g e in the hupA hupB himA triple m u t a n t was similar to that in the hupA hupB d o u b l e m u t a n t (Fig. 1E a n d D, respectively), with
D
B
10000.0 !
looo.o
the s e c o n d step c o m p l e t e l y inhibited. H o w e v e r , w h e n we
1.0 0.1 I 413
i
I 4O
80
80
I
i
4O
tested the i n t r a c e l l u l a r g r o w t h of the T 4 p h a g e in the
J
100
I 4O
8O
hupA hupB hireD triple m u t a n t we f o u n d t h a t the g r o w t h of the p h a g e was h i g h l y i n h i b i t e d e v e n at the first cycle (Fig. I G ) . C o m p l e m e n t a t i o n B0
"rime (min)
F
plasmid
analysis of triple m u t a n t
pPthiphimA-5
revealed
that
the
c u r v e s of i n t r a c e l l u l a r p h a g e g r o w t h in these strains w e r e
100000.0
E
cells u s i n g
G
c o m p a r a b l e to t h a t of T4 in a hupA hupB d o u b l e m u t a n t
H
(Fig. I F , H a n d D, respectively). 1000.0.
T h i s i n d i c a t e s t h a t the H i m D s u b u n i t can f o r m a func-
0 1000~
t i o n a l h o m o d i m e r t h a t is a b l e to s u b s t i t u t e for the H i m D
,o.o
H i m A h e t e r o d i m e r . We i n v o k e the H i m D d i m e r , b e c a u s e Z u l i a n e l l o et al. (1994) h a v e s h o w n t h a t active I H F p r o tein c o u l d be r e c o n s t i t u t e d in v i t r o f r o m the i n d i v i d u a l
01
a,
~0
4o
80
,0
8o
,;
,o
"nine (rain)
ble of specifically r e c o g n i z i n g the s a m e ihf s e q u e n c e , but
Fig. 1. The effect of lack of IHF or HU proteins on the growth of phage T4. Panels show the T4 phage growth in E. coli strains: SD1286 (galK2 galE4 rpsL thi-1 A(lac-pro) KmR), wt (A); SD1287 (himA[ASma] hip/ himD::cat), himA hireD double mutant (B); SD1287, himA hireD double mutant carring pPchiphimA-5 plasmid (C); A5196 (hupA16::kan hupBll::cat), hupA hupB double mutant (D); A5127 (hupA16::kan hupBll::cat himA A82::Tn10), hupA hupB himA triple mutant (E); A5127, hupA hupB himA triple mutant carring pPchiphimA-5 plasmid (F); A5179 (hupA16::kan hupBll::cat hip/himD157::TnlO) hupA hupB hireD triple mutant (G); A5179 hupA hupB himD triple mutant carring pPchiphimA-5 plasmid (H). pfu/C, plaque-forming units/infective center. Methods: Bacteria were grown in Luria Broth (LB) to A575n m = 0.1, sedimented (2000 x g, 10 min) and suspended in 1/10 volume ( 10 ml) of TM/3 mM NaN 3. After 5 min at the 37°C, T4 phage was added at a moi (multiplicity of infection) of 0.05 and incubated for 10 min. Then it was diluted 10-fold in TM/3 mM NaN 3 and sedimented. Bacteria were suspended in 1 ml TM buffer ( 10 mM Tris pH 8.0/10 mM MgSO4) with 3 mM NaN 3. The suspension was then diluted 1000-fold with LB prewarmed to 37°C (time zero) and aerated in a water-bath shaker at this temperature. The number of infective centres was estimated from twelve samples taken during the 5-10-min interval by plating. The mean value is represented as a horizontal line in each figure. The intracellular progeny phages (samples were previously shaken for 1 min with an equal volume of chloroform and cleared by centrifugation) were estimated by plating on E. coil CR63 indicator bacteria. Each experiment was carried out five times for checking the reproducibility; curves were the same in each case. d e v e l o p m e n t i n d i r e c t l y as it d o e s for a n u m b e r of h o s t f u n c t i o n s ( F r e u n d l i c h et al., 1992). I H F specifically b i n d s to a n a s y m m e t r i c c o n s e n s u s s e q u e n c e s w h i c h are u s u a l l y f o u n d in A + T - r i c h r e g i o n s ( K u r et al., 1989). C o m p u t e r analysis o f T 4 D N A
r e v e a l e d m a n y c o n s e n s u s - l i k e ihf
sites. T h i s is n o t surprising, c o n s i d e r i n g the A + T - r i c h n a t u r e of b o t h the T 4 D N A consensus.
(64% A+T)
I H F subunits, a n d t h a t h o m o d i m e r s of H i m D w e r e c a p a -
a n d the ihf
w i t h a b o u t 100-times l o w e r affinity, w h e n c o m p a r e d w i t h the I H F - D N A
complex. The HimA-DNA
c o m p l e x was
less stable a n d was o n l y o b s e r v e d w h e n a large excess of HimA
was used. T h i s c o u l d e x p l a i n o u r results that
H i m D h o m o d i m e r a l o n e is f u n c t i o n a l in vivo.
ACKNOWLEDGEMENTS
T h i s s t u d y was s u p p o r t e d by the M i n i s t r y of N a t i o n a l E d u c a t i o n a n d the State C o m m i t t e e for Scientific R e s e a r c h ( p r o j e c t K B N N o . 4 1308 91 01).
REFERENCES Freundlich, M., Ramani, N., Mathew, E., Sirko, A. and Tsui, P.: The role of integration host factor in gene expression in Escherichia coli. Mol. Microbiol. 6 (1992) 2557-2563. Friedman, D.I.: Integration host factor: a protein for all reasons. Cell 55 (1988) 545-554. Kur, J., Hasan, N. and Szybalski, W.: Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p~ promoter and its mutants. Gene 81 (1989) 1 15. Nash, H.A., Robertson, C.A., Flamm, E., Weisberg, R.A. and Miller, H.I.: Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits. J. Bacteriol. 169 (1987) 4124- 4127. Zulianello, L., de la Gorgue de Rosny, E., Van Ulsen, P., Van de Putte, P. and Goosen, N.: The HimA and HireD subunits of integration host factor can specifically bind to DNA as homodimers. EMBO J. 13 (1994) 1534-1540.
131
GENE 08970
Mutations in H U and IHF affect bacteriophage T4 growth: HimD subunits of IHF appear to function as homodimers (Integration host factor; ihfsite; histone-like protein; one-step growth experiment)
Barbara Zablewska and J6zef Kur Department of Microbiology, Technical University of Gdahsk, 80-952 Gdahsk, Poland Received by J. Wild: 8 October 1994; Revised/Accepted: 16 February 1995; Received at publishers: 7 April 1995
SUMMARY
The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., ~, qb80, Mu and fl) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants (hupA hupB himA and hupA hupB himD) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.
The integration host factor (IHF) of Escherichia coli is a histone-like, heterodimeric protein consisting of the products of the himA gene and the hireD~hip gene. IHF participates in various processes (see, for review, Friedman, 1988). The purpose of the present work was to test (i) whether IHF and HU are needed for coliphage T4 propagation and if so (ii) whether IHF subunits are able to function as homodimers. Absence of HU (using hupA- hupB- as host) had no effect on T4 adsorption and resulted only in a slight inhibition of phage growth (including the eclipse period, as well as the average burst size) during the first cycle of development (compare Fig. 1A and D), but the second cycle of the T4 growth was completely inhibited (Fig. 1D). Correspondence to: Dr. J. Kur, Technical University of Gdafisk, G. Narutowicza Street 11/12, 80-952 Gdafisk, Poland. Tel. (48-58) 472-302; Fax (48-58) 472-694; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); A, deletion; himA, gene encoding the a subunit of IHF; hireD, gene encoding the ~t subunit of IHF; HU, major component of the proteins bound in the E. coli nucleoid; hupA, gene encoding HUa; hupB, gene encoding HUb; IHF, integration host factor; LB, Luria-Bertani (broth); wt, wild type.
SSD! 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 2 5 2 - 9
Next, the T4 phage growth experiment was carried out in the absence of IHF protein using himA himD double mutant. The curves of intracellular phage growth at the first cycle of development in the wt cells and in the himA hireD double mutant cells were similar, except that the eclipse period was about 10 min shorter in wt cells (Fig. 1A,B). In the second cycle, the T4 growth was much lower in the himA himD double mutant cells (over 10-fold reduction) as compared to the wt cells. The adsorption of the T4 phage to the wt strain of E. coli was the same as to the himA himD double mutant (results not shown). These results suggest that IHF might be needed for bacteriophage T4 growth. Since adsorption of T4 was not affected in double mutants, the inhibition of phage growth in the absence of IHF protein must occur in a post-adsorption step. Complementation analysis was carried out to confirm our suggestion. Plasmid pPLhiphimA-5 (Nash et al., 1987) was used to transform himA himD double mutant cells. The curve of intracellular phage growth in this strain (Fig. 1C) was comparable to that of T4 in a wt strain (Fig. 1A). Thus, plasmid producing IHF complemented the defect of himA himD double mutant for T4 phage growth. It has not yet been reported that bacteriophage T4 growth depends on IHF function. IHF could regulate T4
132 100000.0
0
T h e i n t r a c e l l u l a r g r o w t h of the T 4 p h a g e in the hupA hupB himA triple m u t a n t was similar to that in the hupA hupB d o u b l e m u t a n t (Fig. 1E a n d D, respectively), with
D
B
10000.0 !
looo.o
the s e c o n d step c o m p l e t e l y inhibited. H o w e v e r , w h e n we
1.0 0.1 I 413
i
I 4O
80
80
I
i
4O
tested the i n t r a c e l l u l a r g r o w t h of the T 4 p h a g e in the
J
100
I 4O
8O
hupA hupB hireD triple m u t a n t we f o u n d t h a t the g r o w t h of the p h a g e was h i g h l y i n h i b i t e d e v e n at the first cycle (Fig. I G ) . C o m p l e m e n t a t i o n B0
"rime (min)
F
plasmid
analysis of triple m u t a n t
pPthiphimA-5
revealed
that
the
c u r v e s of i n t r a c e l l u l a r p h a g e g r o w t h in these strains w e r e
100000.0
E
cells u s i n g
G
c o m p a r a b l e to t h a t of T4 in a hupA hupB d o u b l e m u t a n t
H
(Fig. I F , H a n d D, respectively). 1000.0.
T h i s i n d i c a t e s t h a t the H i m D s u b u n i t can f o r m a func-
0 1000~
t i o n a l h o m o d i m e r t h a t is a b l e to s u b s t i t u t e for the H i m D
,o.o
H i m A h e t e r o d i m e r . We i n v o k e the H i m D d i m e r , b e c a u s e Z u l i a n e l l o et al. (1994) h a v e s h o w n t h a t active I H F p r o tein c o u l d be r e c o n s t i t u t e d in v i t r o f r o m the i n d i v i d u a l
01
a,
~0
4o
80
,0
8o
,;
,o
"nine (rain)
ble of specifically r e c o g n i z i n g the s a m e ihf s e q u e n c e , but
Fig. 1. The effect of lack of IHF or HU proteins on the growth of phage T4. Panels show the T4 phage growth in E. coli strains: SD1286 (galK2 galE4 rpsL thi-1 A(lac-pro) KmR), wt (A); SD1287 (himA[ASma] hip/ himD::cat), himA hireD double mutant (B); SD1287, himA hireD double mutant carring pPchiphimA-5 plasmid (C); A5196 (hupA16::kan hupBll::cat), hupA hupB double mutant (D); A5127 (hupA16::kan hupBll::cat himA A82::Tn10), hupA hupB himA triple mutant (E); A5127, hupA hupB himA triple mutant carring pPchiphimA-5 plasmid (F); A5179 (hupA16::kan hupBll::cat hip/himD157::TnlO) hupA hupB hireD triple mutant (G); A5179 hupA hupB himD triple mutant carring pPchiphimA-5 plasmid (H). pfu/C, plaque-forming units/infective center. Methods: Bacteria were grown in Luria Broth (LB) to A575n m = 0.1, sedimented (2000 x g, 10 min) and suspended in 1/10 volume ( 10 ml) of TM/3 mM NaN 3. After 5 min at the 37°C, T4 phage was added at a moi (multiplicity of infection) of 0.05 and incubated for 10 min. Then it was diluted 10-fold in TM/3 mM NaN 3 and sedimented. Bacteria were suspended in 1 ml TM buffer ( 10 mM Tris pH 8.0/10 mM MgSO4) with 3 mM NaN 3. The suspension was then diluted 1000-fold with LB prewarmed to 37°C (time zero) and aerated in a water-bath shaker at this temperature. The number of infective centres was estimated from twelve samples taken during the 5-10-min interval by plating. The mean value is represented as a horizontal line in each figure. The intracellular progeny phages (samples were previously shaken for 1 min with an equal volume of chloroform and cleared by centrifugation) were estimated by plating on E. coil CR63 indicator bacteria. Each experiment was carried out five times for checking the reproducibility; curves were the same in each case. d e v e l o p m e n t i n d i r e c t l y as it d o e s for a n u m b e r of h o s t f u n c t i o n s ( F r e u n d l i c h et al., 1992). I H F specifically b i n d s to a n a s y m m e t r i c c o n s e n s u s s e q u e n c e s w h i c h are u s u a l l y f o u n d in A + T - r i c h r e g i o n s ( K u r et al., 1989). C o m p u t e r analysis o f T 4 D N A
r e v e a l e d m a n y c o n s e n s u s - l i k e ihf
sites. T h i s is n o t surprising, c o n s i d e r i n g the A + T - r i c h n a t u r e of b o t h the T 4 D N A consensus.
(64% A+T)
I H F subunits, a n d t h a t h o m o d i m e r s of H i m D w e r e c a p a -
a n d the ihf
w i t h a b o u t 100-times l o w e r affinity, w h e n c o m p a r e d w i t h the I H F - D N A
complex. The HimA-DNA
c o m p l e x was
less stable a n d was o n l y o b s e r v e d w h e n a large excess of HimA
was used. T h i s c o u l d e x p l a i n o u r results that
H i m D h o m o d i m e r a l o n e is f u n c t i o n a l in vivo.
ACKNOWLEDGEMENTS
T h i s s t u d y was s u p p o r t e d by the M i n i s t r y of N a t i o n a l E d u c a t i o n a n d the State C o m m i t t e e for Scientific R e s e a r c h ( p r o j e c t K B N N o . 4 1308 91 01).
REFERENCES Freundlich, M., Ramani, N., Mathew, E., Sirko, A. and Tsui, P.: The role of integration host factor in gene expression in Escherichia coli. Mol. Microbiol. 6 (1992) 2557-2563. Friedman, D.I.: Integration host factor: a protein for all reasons. Cell 55 (1988) 545-554. Kur, J., Hasan, N. and Szybalski, W.: Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p~ promoter and its mutants. Gene 81 (1989) 1 15. Nash, H.A., Robertson, C.A., Flamm, E., Weisberg, R.A. and Miller, H.I.: Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits. J. Bacteriol. 169 (1987) 4124- 4127. Zulianello, L., de la Gorgue de Rosny, E., Van Ulsen, P., Van de Putte, P. and Goosen, N.: The HimA and HireD subunits of integration host factor can specifically bind to DNA as homodimers. EMBO J. 13 (1994) 1534-1540.