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Recognition of cell surface receptors is controlled by invertible DNA of phage Mu
FEMS Microbiology Letters 16 (1983) 131-135 Published by Elsevier Biomedical Press
131
Recognition of cell surface receptors is controlled by invertible D N A of phage Mu D i e t m a r K a m p and R o d i c a S a n d u l a c h e Max-Planck-lnstitut fiir Biochemie, 8033 Martinsried bei Miinchen, F.R.G.
Received and accepted 13 July 1982
1. I N T R O D U C T I O N Inversion of the G segment of bacteriophage Mu DNA is one of the few examples in prokaryotes where the expression of genes is controlled through a rearrangement of DNA. Production of phage particles which form plaques on Escherichia coli K12 requires the G segment to be in the G ( + ) orientation [1,2] whereas defective phage particles are obtained from a Mu G ( - ) lysogen. Several additional results have indicated that the G segment encodes functions which are involved in the adsorption of phage and the injection of its DNA into the bacterial cell [1,3-5]. In this communication we present evidence that Mu G ( + ) and G ( - ) phage particles recognize different receptors in the cell wall of Gram-negative bacteria and as a consequence infect different hosts: Mu G ( - ) , but not Mu G ( + ) , can be propagated on Enterobacter cloacae, Serratia marcescens [6]. Other hosts for Mu G ( - - ) phage are Citrobacter freundii [7] and Erwinia carotovora (A. Toussaint, personal communication). The biological significance of the invertible segment lies therefore in the extension of the host range of the phage, raising the possibility that Mu might persist in other environments, since some of the newly found hosts like Serratia differ in their habitat from E. coli.
2. MATERIALS AND METHODS The following bacterial strains were used: E. co# KI2 C600 [8], E. cloacae [9], S. marcescens S b [10], S. kiliensis ATCC7462, E. aerogenes ATCC13048, Proteus vulgaris ATCC9820, Agrobacterium tumefaciens C583 (obtained from K. Geider), the core type strains E. coli C, F576,
F635,
F2556
from
the
Freiburg
collection,
Salmonella arizonae hinshawii from the collection
of the Max von Pettenkofer Institute, Munich. Mu gin +, Mu gin4454 G ( + ) and Mu gin~45_s G ( - ) lysates were made by heat induction from DK394, DK1066 and DK947 [1]. Bacteria were grown at 37°C in YT medium to saturation and plated with soft agar on YT plates [11]. Phage lysates were diluted in TM (0.01 M Tris-HC1 pH 7.8, 0.01 M MgSO4) and 10-btl aliquots were spotted on the bacterial lawn. Plates were incubated overnight at 40°C. Lipopolysaccharide was prepared from Salmonella and E. co# strains according to the procedure of Galanos [12] and from the other strains according to the method of Westphal [13]. Irreversible binding of LPS to phage was tested by mixing 0.1 ml phage lysate (5.103 p f u / m l ) with 0.1 ml LPS (5 mg~ml). After incubation for 1 h at 37°C, 0.2 ml indicator (E. co# K12 C600 for Mu G ( + ) , E. cloacae for Mu G ( - ) and E. co# C for Mu G ( - ) hl01) was added. The mixture was plated with soft agar on YT agar and incubated overnight at 42°C.
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies
132
3. RESULTS
ence of a host-specifi'ed restriction system which restricts infecting Mu DNA. When grown on these hosts, Mu plated with a 100-fold higher efficiency due to the apparent modification of its DNA.
3.1. Host range o f M u G( + ) and G( - )
Rapid screening for specific plating of Mu G( + ) or G ( - ) phage on bacterial strains has become possible through the existence of a mutant strain Mu gin445_5. This mutant is defective for a sitespecific recombination function (Gin) which is required for inversion of the G segment. High-titer lysates of Mu G ( + ) or G ( - ) can be thus obtained after heat induction of the appropriate lysogenic bacterium [1]. When we tested such lysates with various enterobacterial strains, we observed that Mu gin G ( - ) but not Mu G ( + ) phage formed plaques on E. cloacae and S. marcescens S b. This was also consistent with the plating efficiency of Mu gin ÷ (Table 1). A Mu gin ÷ lysate which had been prepared by heat induction of a lysogen contained about the same number of G ( + ) and G ( - ) particles and plated with about the same efficiency on S. marcescens and E. cloacae as Mu gin G ( - ) . In contrast, lysates which had been made by serial infection of E. coli C600 and which contained 99% G ( + ) phage, plated with a lower efficiency on hosts that are sensitive for Mu G ( - ) . The overall lower plating efficiency on S. marcescens and E. cloacae can be explained by the pres-
3.2. Abortive infection o f M u G ( - )
On A. tumefaciens, P. vulgaris, Serratia kiliensis and Enterobacter aerogenes Mu G( - ) did not make plaques but the undiluted lysate inhibited bacterial growth as seen by clearing of the bacterial lawn, whereas a concentrated lysate of Mu G( + ) did not give the same effect. This showed that the inhibitory effect was promoted by specific interaction of Mu G ( - ) phage particles with these bacteria. Mu obviously does not replicate in these organisms which is consistent with the low yield of plaqueforming phage upon heat induction, when Mu is introduced into A. tumefaciens via an episome [14]. This effect is reminiscent of the abortive infection of phage P1 in Serratia and Pseudomonas aeruginosa [15]. 3.3. Lipopolysaccharide as receptor for M u G( + ) and M u G( -- ) phage
We observed a loss of infectivity when lipopolysaccharide (LPS) of E. coli C600 was mixed with
Table 1 Plating efficiency of Mu G( + ) and Mu G( - ) Except for Mu vir. phage was propagated by heat induction of a lysogenic bacterium. The relative plating efficiency of Mu G ( - ) grown by heat induction of an E. coli K12 lysogen was calculated as the ratio of plaque-forming units to phage particles. The n u m b e r of phage particles was estimated as follows. Since lysogens of Mu G ( + ) and Mu G( ) produce about the same n u m b e r of phage particles after heat induction [1 l, Mu G ( + ) and Mu G ( - - ) lysates were made side-by-side and the n u m b e r of G ( - ) phage particles was set equal to the n u m b e r of plaque-forming units obtained for the Mu G ( + ) lysate. For plating of lysates prepared from an E. cloacae host a h s d R derivative of C600 was used. Host
E. E. E. E. E. E.
coil K I 2 cofiKl2 coli K12 coliKl2 cloacae cloacae
Phage
Mu cts62 gin445. 5 Mucts62gin~5. 5 Mu cts62 gin +
Muvir Mu cts62 g i n , s _ 5 Mu cts62 gin +
Orientation of the G segment
Relative plating efficiency on E. coli K12
E. cloacae
S. marcescens
G(+) G(-) G(+)+G(-) G(+)>>G(-) G(--) G(+)+G(-)
1 <10 8 1 1 < 10 7 1
<10 ~s 10 3 2-10 4 <10 7 1 1
<10 8 5.10 5.10 <10 7 2.10 10 2
3 3 2
133 Table 2 Irreversible binding of Mu G( + ) and Mu G ( - ) by phage to purified lipopolysaccharide Relative efficiency of plating after incubation with LPS of E. E. S. P. A. E.
coli K 12 C600 cloacae marcescens vulgaris turnefaciens coli C
Mu G ( + )
Mu G(
0.004 1 1.1 1 1.2 1
1 0.005 0.002 0.002 0.22 1
)
Mu G ( - ) hl01
1 0.01 n.d. n.d. n.d. <0.001
n.d., not determined.
Mu G ( + ) phage. The receptor for Mu G ( + ) phage could therefore be identified as part of the LPS. This suggested a possible explanation for the different host range of Mu G ( - ) , namely that Mu G ( + ) and G(--) phage recognize different cell wall receptors. Tests with purified cell walls
(Table2) proved this notion to be correct: Mu G ( - ) was inactivated by LPS from E. cloacae and not by LPS from E. coli K12, whereas plating of Mu G ( + ) phage was unaffected by LPS from E. cloacae. The strong interaction of Mu G ( - ) with purified LPS from A. tumefaciens and P. vulgaris
Table 3 Mu-sensitive enterobacterial strains with different lipopolysaccharide core types LPS core type
Structure a
Ra
~
Phage sensitivity
GIcNAc Salmonella arizonae
R2 E. coli 0100
E. coli K 12
Gal
1,2
a1,2
Glc
a1,3
Gal
Hep
ot 1,6,3 Glc
1,7/31,3 Hep--
Hep--(KDO)3-
GIcNAc Gal Hep a ~l1,2 a 1,6 1,7 I al,2 al,3 al,3 1,3 Hep--(KDO)3Glc Glc Glc Hep Gal GlcNAc a 1,6 ,B] 1,6 al,3 al,3 1,3 I al,2 I Glc Glc He,p - - Hep--(KDO)3Glc 7',, 1,,'7
Mu G ( + )
Mu G ( + )
Mu G ( + )
hip 1,7 Hep a1,2 Gal R 1 E. coli C
Gal ill,3
Glc
Hep al,3,
GIc
a The core structures were taken from [16].
al,3
GIc - - Hep
1,3 Hep--(KDO)3-
Mu G ( - - ) h
134 showed that the inability to form plaques on these hosts was not due to poor interaction of the phage with the cell wall, but must be caused by an intracellular block of phage multiplication. 3.4. Core type specificity of G( + ) and G( - ) phage We tested whether E. coli strains which are representatives of different lipopolysaccharide core types can be infected by Mu G ( + ) or Mu G ( - ) (Table 3). We found that core type R2 contains a receptor for Mu G ( + ) . R2 is very similar to the core of K 12. Mu G ( - ) plates with a frequency of 10-7 on the core type R1 strain E. coli C in agreement with [7]. Since E. coli C does not restrict foreign D N A [17], the low plating efficiency suggested to us that only host-range mutants of Mu G ( - ) plated on E. coli C. This was substantiated by experiments with purified LPS of E. coli C. Mu G ( - ) phage was not inactivated by LPS, but infectivity of the mutant phage Mu G ( - ) h l 0 1 was completely blocked. The R1 core type therefore is not identical, but similar to the structure that is recognized by Mu G ( - ) . The core type Ra found in Salmonella differs from core type R2 in only one position. Although in our initial screening we did not find plaques of Mu G ( + ) on S. arizonae, this is probably due to the strong effect of hostmediated restriction of non-modified Mu D N A , since Mu G ( + ) morn + can obviously overcome the restriction to some extent and make plaques on S. arizonae.
of every Gram-negative species will be a potential host for Mu. In some cases phage adsorption is not the limiting factor for the host range because Mu adsorbs to and penetrates the cell wall but is unable to multiply in these bacteria. It remains to be seen which functions of Mu are expressed in such abortive infections and whether Mu can for instance persist as a transposon. As Mu can grow in bacteria which differ in their habitat from E. coli, we would expect to find Mu or Mu-like phage in soil or other environments. Interesting in this context is that Mu-like phage have been isolated from Pseudomonas [ 18,19]. These results not only shed light on the physiological role of the invertible G segment for the life cycle of Mu, but they also have a twofold practical importance. In recent years Mu has become a widely used tool for bacterial geneticists. One can use Mu, or manipulated Mu genomes, to transduce, fuse or rearrange bacterial D N A at will [20-22]. This technology will become available for other bacteria, the genetics of which are less developed. Since Mu is also considered a giant transposable element studies of the mechanism of transposition can be extended to other bacterial species. ACKNOWLEDGEMENTS The initial part of this work was carried out at the Cold Spring H a r b o r Laboratory and was supported by N I H grant GM23996 to Louise T. Chow. We thank Phyllis Myers and Klaus Hantke for supplying us with bacterial strains.
4. C O N C L U S I O N S The invertible G segment provides phage Mu with the opportunity of infecting m a n y more bacteria than usual. The orientation of the G segment controls the expression of two sets of functions which interact specifically with different receptors in the outer m e m b r a n e of Gram-negative bacteria. It follows that these gene products which confer the host specificity must be components of the phage tail and, in analogy to other phage, are probably the tail fibers. Because of the variability of the lipopolysaccharide structure in the bacterial kingdom, it is very likely that at least one member
REFERENCES [1] Kamp, D., Kahmann, R., Zipser, D., Broker, T.R. and Chow, L. (1978) Nature 271,577-580. [2] Symonds, N. and Coelho, A. (1978) Nature 271, 573-574. [3] Bukhari, A.I. and Ambrosio, L. (1978) Nature 271, 575577. [4l Toussaint, A., Lefebre, N., Scott, J.R., Cowan, J.A., Bruijn, F. and Bukhari, A.I. (1978) Virology 89, 146-161. [5] Howe, M.M., Schumm, J.W. and Taylor, A.L. (1979) Virology 92, 108-124. [6] Kamp, D. (1981) in Microbiology- 1981 (Schlessinger, D., Ed.), pp. 73-76. Am. Soc. Microb., Washington, DC. [7] Van de Putte, P., Cramer, S. and Giphart-Gassler, M. (1980) Nature 286, 218-222.
135 [8] Appleyard, R.K. (1954) Genetics 39, 429-435. [9] Hartmann, H. and Goebel, W. (1977) FEBS Lett., 80, 285-287. [10] Endow, S.A. and Roberts, R.J. (1977) J. Mol. Biol. 112, 521-529. [11] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. [12] Galanos, Ch., Ltideritz, O. and Westphal, O. (1969) Eur. J. Biochem. 9, 245-249. [13] Westphal, O. and Javin, R. (1965) in Bacterial Lipopolysaccharides, Methods in Carbohydrate Research (Whistler, R.C. and Wolform, M.L., Eds.), Vol. V, pp. 81-91. Academic Press, New York. [14] Van Vliet, F., Silva, B., Van Montagu, M. and Schell, J. (1978) Plasmid 1(4), 446-455.
[15] Amati, P. (1962) J. Bacteriol. 83, 433-434. [16] Jansson, P.E., Lindberg, A.A., Lindberg, B. and Wollin, R. (1980) Eur. J. Biochem. 115, 571-577. [17] Bertani, G. and Weigle, J.J. (1953) J. Bacteriol. 65, 113. [18] Krylov, V.N., Bogush, V.G., Yanenko, A.S. and Kirsanov, N.B. (1980) Genetika 16(6), 975-984. [19] Krylov, V.N., Bogush, V.G. and Shapiro, J. (1980) Genetika 15(5), 824-832. [20] Faelen, M. and Toussaint, A. (1976) J. Mol. Biol. 104, 525-539. [21] Faelen, M.A., Toussaint, A. and Resibois, A. (1979) Mol. Gen. Genet. 176, 191-197. [22] Casadaban, M.J. and Cohen, S.N. (1979) Proc. Natl. Acad. Sci. USA 76, 9, 4530-4533.
131
Recognition of cell surface receptors is controlled by invertible D N A of phage Mu D i e t m a r K a m p and R o d i c a S a n d u l a c h e Max-Planck-lnstitut fiir Biochemie, 8033 Martinsried bei Miinchen, F.R.G.
Received and accepted 13 July 1982
1. I N T R O D U C T I O N Inversion of the G segment of bacteriophage Mu DNA is one of the few examples in prokaryotes where the expression of genes is controlled through a rearrangement of DNA. Production of phage particles which form plaques on Escherichia coli K12 requires the G segment to be in the G ( + ) orientation [1,2] whereas defective phage particles are obtained from a Mu G ( - ) lysogen. Several additional results have indicated that the G segment encodes functions which are involved in the adsorption of phage and the injection of its DNA into the bacterial cell [1,3-5]. In this communication we present evidence that Mu G ( + ) and G ( - ) phage particles recognize different receptors in the cell wall of Gram-negative bacteria and as a consequence infect different hosts: Mu G ( - ) , but not Mu G ( + ) , can be propagated on Enterobacter cloacae, Serratia marcescens [6]. Other hosts for Mu G ( - - ) phage are Citrobacter freundii [7] and Erwinia carotovora (A. Toussaint, personal communication). The biological significance of the invertible segment lies therefore in the extension of the host range of the phage, raising the possibility that Mu might persist in other environments, since some of the newly found hosts like Serratia differ in their habitat from E. coli.
2. MATERIALS AND METHODS The following bacterial strains were used: E. co# KI2 C600 [8], E. cloacae [9], S. marcescens S b [10], S. kiliensis ATCC7462, E. aerogenes ATCC13048, Proteus vulgaris ATCC9820, Agrobacterium tumefaciens C583 (obtained from K. Geider), the core type strains E. coli C, F576,
F635,
F2556
from
the
Freiburg
collection,
Salmonella arizonae hinshawii from the collection
of the Max von Pettenkofer Institute, Munich. Mu gin +, Mu gin4454 G ( + ) and Mu gin~45_s G ( - ) lysates were made by heat induction from DK394, DK1066 and DK947 [1]. Bacteria were grown at 37°C in YT medium to saturation and plated with soft agar on YT plates [11]. Phage lysates were diluted in TM (0.01 M Tris-HC1 pH 7.8, 0.01 M MgSO4) and 10-btl aliquots were spotted on the bacterial lawn. Plates were incubated overnight at 40°C. Lipopolysaccharide was prepared from Salmonella and E. co# strains according to the procedure of Galanos [12] and from the other strains according to the method of Westphal [13]. Irreversible binding of LPS to phage was tested by mixing 0.1 ml phage lysate (5.103 p f u / m l ) with 0.1 ml LPS (5 mg~ml). After incubation for 1 h at 37°C, 0.2 ml indicator (E. co# K12 C600 for Mu G ( + ) , E. cloacae for Mu G ( - ) and E. co# C for Mu G ( - ) hl01) was added. The mixture was plated with soft agar on YT agar and incubated overnight at 42°C.
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies
132
3. RESULTS
ence of a host-specifi'ed restriction system which restricts infecting Mu DNA. When grown on these hosts, Mu plated with a 100-fold higher efficiency due to the apparent modification of its DNA.
3.1. Host range o f M u G( + ) and G( - )
Rapid screening for specific plating of Mu G( + ) or G ( - ) phage on bacterial strains has become possible through the existence of a mutant strain Mu gin445_5. This mutant is defective for a sitespecific recombination function (Gin) which is required for inversion of the G segment. High-titer lysates of Mu G ( + ) or G ( - ) can be thus obtained after heat induction of the appropriate lysogenic bacterium [1]. When we tested such lysates with various enterobacterial strains, we observed that Mu gin G ( - ) but not Mu G ( + ) phage formed plaques on E. cloacae and S. marcescens S b. This was also consistent with the plating efficiency of Mu gin ÷ (Table 1). A Mu gin ÷ lysate which had been prepared by heat induction of a lysogen contained about the same number of G ( + ) and G ( - ) particles and plated with about the same efficiency on S. marcescens and E. cloacae as Mu gin G ( - ) . In contrast, lysates which had been made by serial infection of E. coli C600 and which contained 99% G ( + ) phage, plated with a lower efficiency on hosts that are sensitive for Mu G ( - ) . The overall lower plating efficiency on S. marcescens and E. cloacae can be explained by the pres-
3.2. Abortive infection o f M u G ( - )
On A. tumefaciens, P. vulgaris, Serratia kiliensis and Enterobacter aerogenes Mu G( - ) did not make plaques but the undiluted lysate inhibited bacterial growth as seen by clearing of the bacterial lawn, whereas a concentrated lysate of Mu G( + ) did not give the same effect. This showed that the inhibitory effect was promoted by specific interaction of Mu G ( - ) phage particles with these bacteria. Mu obviously does not replicate in these organisms which is consistent with the low yield of plaqueforming phage upon heat induction, when Mu is introduced into A. tumefaciens via an episome [14]. This effect is reminiscent of the abortive infection of phage P1 in Serratia and Pseudomonas aeruginosa [15]. 3.3. Lipopolysaccharide as receptor for M u G( + ) and M u G( -- ) phage
We observed a loss of infectivity when lipopolysaccharide (LPS) of E. coli C600 was mixed with
Table 1 Plating efficiency of Mu G( + ) and Mu G( - ) Except for Mu vir. phage was propagated by heat induction of a lysogenic bacterium. The relative plating efficiency of Mu G ( - ) grown by heat induction of an E. coli K12 lysogen was calculated as the ratio of plaque-forming units to phage particles. The n u m b e r of phage particles was estimated as follows. Since lysogens of Mu G ( + ) and Mu G( ) produce about the same n u m b e r of phage particles after heat induction [1 l, Mu G ( + ) and Mu G ( - - ) lysates were made side-by-side and the n u m b e r of G ( - ) phage particles was set equal to the n u m b e r of plaque-forming units obtained for the Mu G ( + ) lysate. For plating of lysates prepared from an E. cloacae host a h s d R derivative of C600 was used. Host
E. E. E. E. E. E.
coil K I 2 cofiKl2 coli K12 coliKl2 cloacae cloacae
Phage
Mu cts62 gin445. 5 Mucts62gin~5. 5 Mu cts62 gin +
Muvir Mu cts62 g i n , s _ 5 Mu cts62 gin +
Orientation of the G segment
Relative plating efficiency on E. coli K12
E. cloacae
S. marcescens
G(+) G(-) G(+)+G(-) G(+)>>G(-) G(--) G(+)+G(-)
1 <10 8 1 1 < 10 7 1
<10 ~s 10 3 2-10 4 <10 7 1 1
<10 8 5.10 5.10 <10 7 2.10 10 2
3 3 2
133 Table 2 Irreversible binding of Mu G( + ) and Mu G ( - ) by phage to purified lipopolysaccharide Relative efficiency of plating after incubation with LPS of E. E. S. P. A. E.
coli K 12 C600 cloacae marcescens vulgaris turnefaciens coli C
Mu G ( + )
Mu G(
0.004 1 1.1 1 1.2 1
1 0.005 0.002 0.002 0.22 1
)
Mu G ( - ) hl01
1 0.01 n.d. n.d. n.d. <0.001
n.d., not determined.
Mu G ( + ) phage. The receptor for Mu G ( + ) phage could therefore be identified as part of the LPS. This suggested a possible explanation for the different host range of Mu G ( - ) , namely that Mu G ( + ) and G(--) phage recognize different cell wall receptors. Tests with purified cell walls
(Table2) proved this notion to be correct: Mu G ( - ) was inactivated by LPS from E. cloacae and not by LPS from E. coli K12, whereas plating of Mu G ( + ) phage was unaffected by LPS from E. cloacae. The strong interaction of Mu G ( - ) with purified LPS from A. tumefaciens and P. vulgaris
Table 3 Mu-sensitive enterobacterial strains with different lipopolysaccharide core types LPS core type
Structure a
Ra
~
Phage sensitivity
GIcNAc Salmonella arizonae
R2 E. coli 0100
E. coli K 12
Gal
1,2
a1,2
Glc
a1,3
Gal
Hep
ot 1,6,3 Glc
1,7/31,3 Hep--
Hep--(KDO)3-
GIcNAc Gal Hep a ~l1,2 a 1,6 1,7 I al,2 al,3 al,3 1,3 Hep--(KDO)3Glc Glc Glc Hep Gal GlcNAc a 1,6 ,B] 1,6 al,3 al,3 1,3 I al,2 I Glc Glc He,p - - Hep--(KDO)3Glc 7',, 1,,'7
Mu G ( + )
Mu G ( + )
Mu G ( + )
hip 1,7 Hep a1,2 Gal R 1 E. coli C
Gal ill,3
Glc
Hep al,3,
GIc
a The core structures were taken from [16].
al,3
GIc - - Hep
1,3 Hep--(KDO)3-
Mu G ( - - ) h
134 showed that the inability to form plaques on these hosts was not due to poor interaction of the phage with the cell wall, but must be caused by an intracellular block of phage multiplication. 3.4. Core type specificity of G( + ) and G( - ) phage We tested whether E. coli strains which are representatives of different lipopolysaccharide core types can be infected by Mu G ( + ) or Mu G ( - ) (Table 3). We found that core type R2 contains a receptor for Mu G ( + ) . R2 is very similar to the core of K 12. Mu G ( - ) plates with a frequency of 10-7 on the core type R1 strain E. coli C in agreement with [7]. Since E. coli C does not restrict foreign D N A [17], the low plating efficiency suggested to us that only host-range mutants of Mu G ( - ) plated on E. coli C. This was substantiated by experiments with purified LPS of E. coli C. Mu G ( - ) phage was not inactivated by LPS, but infectivity of the mutant phage Mu G ( - ) h l 0 1 was completely blocked. The R1 core type therefore is not identical, but similar to the structure that is recognized by Mu G ( - ) . The core type Ra found in Salmonella differs from core type R2 in only one position. Although in our initial screening we did not find plaques of Mu G ( + ) on S. arizonae, this is probably due to the strong effect of hostmediated restriction of non-modified Mu D N A , since Mu G ( + ) morn + can obviously overcome the restriction to some extent and make plaques on S. arizonae.
of every Gram-negative species will be a potential host for Mu. In some cases phage adsorption is not the limiting factor for the host range because Mu adsorbs to and penetrates the cell wall but is unable to multiply in these bacteria. It remains to be seen which functions of Mu are expressed in such abortive infections and whether Mu can for instance persist as a transposon. As Mu can grow in bacteria which differ in their habitat from E. coli, we would expect to find Mu or Mu-like phage in soil or other environments. Interesting in this context is that Mu-like phage have been isolated from Pseudomonas [ 18,19]. These results not only shed light on the physiological role of the invertible G segment for the life cycle of Mu, but they also have a twofold practical importance. In recent years Mu has become a widely used tool for bacterial geneticists. One can use Mu, or manipulated Mu genomes, to transduce, fuse or rearrange bacterial D N A at will [20-22]. This technology will become available for other bacteria, the genetics of which are less developed. Since Mu is also considered a giant transposable element studies of the mechanism of transposition can be extended to other bacterial species. ACKNOWLEDGEMENTS The initial part of this work was carried out at the Cold Spring H a r b o r Laboratory and was supported by N I H grant GM23996 to Louise T. Chow. We thank Phyllis Myers and Klaus Hantke for supplying us with bacterial strains.
4. C O N C L U S I O N S The invertible G segment provides phage Mu with the opportunity of infecting m a n y more bacteria than usual. The orientation of the G segment controls the expression of two sets of functions which interact specifically with different receptors in the outer m e m b r a n e of Gram-negative bacteria. It follows that these gene products which confer the host specificity must be components of the phage tail and, in analogy to other phage, are probably the tail fibers. Because of the variability of the lipopolysaccharide structure in the bacterial kingdom, it is very likely that at least one member
REFERENCES [1] Kamp, D., Kahmann, R., Zipser, D., Broker, T.R. and Chow, L. (1978) Nature 271,577-580. [2] Symonds, N. and Coelho, A. (1978) Nature 271, 573-574. [3] Bukhari, A.I. and Ambrosio, L. (1978) Nature 271, 575577. [4l Toussaint, A., Lefebre, N., Scott, J.R., Cowan, J.A., Bruijn, F. and Bukhari, A.I. (1978) Virology 89, 146-161. [5] Howe, M.M., Schumm, J.W. and Taylor, A.L. (1979) Virology 92, 108-124. [6] Kamp, D. (1981) in Microbiology- 1981 (Schlessinger, D., Ed.), pp. 73-76. Am. Soc. Microb., Washington, DC. [7] Van de Putte, P., Cramer, S. and Giphart-Gassler, M. (1980) Nature 286, 218-222.
135 [8] Appleyard, R.K. (1954) Genetics 39, 429-435. [9] Hartmann, H. and Goebel, W. (1977) FEBS Lett., 80, 285-287. [10] Endow, S.A. and Roberts, R.J. (1977) J. Mol. Biol. 112, 521-529. [11] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. [12] Galanos, Ch., Ltideritz, O. and Westphal, O. (1969) Eur. J. Biochem. 9, 245-249. [13] Westphal, O. and Javin, R. (1965) in Bacterial Lipopolysaccharides, Methods in Carbohydrate Research (Whistler, R.C. and Wolform, M.L., Eds.), Vol. V, pp. 81-91. Academic Press, New York. [14] Van Vliet, F., Silva, B., Van Montagu, M. and Schell, J. (1978) Plasmid 1(4), 446-455.
[15] Amati, P. (1962) J. Bacteriol. 83, 433-434. [16] Jansson, P.E., Lindberg, A.A., Lindberg, B. and Wollin, R. (1980) Eur. J. Biochem. 115, 571-577. [17] Bertani, G. and Weigle, J.J. (1953) J. Bacteriol. 65, 113. [18] Krylov, V.N., Bogush, V.G., Yanenko, A.S. and Kirsanov, N.B. (1980) Genetika 16(6), 975-984. [19] Krylov, V.N., Bogush, V.G. and Shapiro, J. (1980) Genetika 15(5), 824-832. [20] Faelen, M. and Toussaint, A. (1976) J. Mol. Biol. 104, 525-539. [21] Faelen, M.A., Toussaint, A. and Resibois, A. (1979) Mol. Gen. Genet. 176, 191-197. [22] Casadaban, M.J. and Cohen, S.N. (1979) Proc. Natl. Acad. Sci. USA 76, 9, 4530-4533.