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Derepressed plasmids of incompatibility group I1 determine two different morphological forms of pilus
PL.ASMlD
9, 331-334 (1983)
Derepressed Plasmids of Incompatibility Group I, Determine Two Different Morphological Forms of Pilus DAVID E. BRADLEY Faculty of Medicine. Memorial University of Newfoundland, St. JohnS, Newfoundland AlB 3V6, Canada Received November 2, 1982 IncI, plasmids R144drd3 and R64drdll determined thick, probably rigid pili in addition to the well-known thinner I, pili. Electron microscopy revealed that the thick I, pili often had large knobs at one end and were pointed at the other. They did not appear to act as receptors for any I-specific bacteriophages.
Bacterial drug resistance plasmids specifying sensitivity to the filamentous bacteriophage If1 (9) were originally classified into severalincompatibility groups forming the socalled I complex (for a listing, see Ref. (2)). However, they have since been tentatively rearranged into only two groups, IncI, and IncI, (6). Receptors for phage If1 and the similar isolate PR64FS (5) are believed to be the serologically distinct thin flexible pili determined by IncI, and IncIz plasmids (5,10), called thin I1 and thin I2 pili, respectively. IncB and IncK plasmids also determine morphologically similar thin flexible pili (2) which are probably not If1 receptors since theseplasmids do not specify sensitivity to If 1. Recently Bradley and Coetzee(3) found that IncIz plasmids determined thick pili as well (thick 12 pili). They were morphologically distinct from thin IZ pili. They acted as receptors for the lipid-containing phage PR4 (12) but not for If1 or PR64FS. In this communication the results of a successfulsearch for thick pili determined by IncI, plasmids (thick I, pili) are described. Escherichia coli 553 (pro met) was used as plasmid host. The IncI, plasmids were R 144drd3 (specifies kanamycin resistance) and R64drdll (specifies streptomycin and tetracycline resistances), both being kindly supplied by N. Datta (see Ref. (II)). Phage PR64FS was kindly donated by J. N. Coetzee.
Brain heart infusion agar (BBL) was used for bacterial growth at 37°C throughout. Drug concentrations were kanamycin, 100 &ml, and tetracycline, 25 clg/ml. Pili were mounted for electron microscopy as described before (2), bacteria being incubated overnight on plates containing appropriate drugs. Phage plus pilus mixtures were prepared as previously described (3). Phage PR64FS was labeled with antibodies for immune electron microscopy by the grid-labeling method (7). Antiserum was raised in rabbits by standard methods (kindly supplied by J. N. Coetzee).All specimens were negatively stained with a mixture of equal volumes of 2% sodium phosphotungstate and 0.1 M ammonium acetate solutions. The discovery that IncIz plasmids determined two distinct morphological types of pilus, thin I2 and thick I2 pili, suggestedthat a similar situation might exist for IncI, plasmids. Two transfer-derepressed IncI, plasmids have been found to determine thick rods. Thesewere morphologically distinct from thin I, pili and appeared different from thick Iz pili in the electron miscoscope. Strain J53(R144drd3) synthesized very many rods with large terminal knobs (Fig. 1). They were approximately 10.5 nm thick (depending on negative staining conditions) compared with the 6 nm of thin I, pili which formed aggregates (Fig. 2). A few Type I pili (7 nm thick)
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0147-619X/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All ngbts of reprcduction in any form reserved
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FIG. 1. Thick I, pilus (longer than average) from an overnight plate of J53(Rl44&3).
Bar = 100 nm.
FIG. 2. The same plate used in Fig. 1, showing a thick I, pilus with a broken knob, and parts of thin 1, pilus aggregates.Bar = 100 nm.
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were present, but these did not aggregate(not illustrated). The knob on the pilus in Fig. 2 seemsto be disintegrating and its appearance suggeststhat it is some form of membranous structure similar to those found on F pili (8), F0 lac pili (4), and D pili (I). Not all thick 11 pili had knobs, which were believed to be located at the proximal end of the pilus like the similar structures just mentioned. The distal tips of the rods were pointed (best seenin Fig. 3). The IncIr plasmid R64drdll also determined thick pili with knobs, but in smaller numbers. Figure 4 illustrates a knob smaller than those shown for R144drd3, but there was considerable size variation with both plasmids. A probable thin I, pilus, or perhaps a Type I pilus, is lying beside the thick one, emphasizing the large difference in thickness. While the thick pili with knobs superficially resemble tailed bacteriophages, they are not bacteriophages since phages have heads of uniform size and tails of constant length. The appearance of the thick pili of both plasmids was consistent with a rigid form (2). Using identical specimen preparation methods, no thick pili could be found for strain 553 without a plasmid (continuous search in the electron microscope for 15 min). Improved electron microscope specimen preparation techniques are undoubtedly the reason why thick II and I2 pili have only been found recently. The distinguishing feature of I complex plasmids is that they support the multiplication of the serologically related filamentous phages If1 and PR64FS. Both adsorb to the tips of conjugative I pili, but none of the published electron micrographs reliably indicates whether these are thick or thin I, or I2 pili, which had not, of course, been distinguished at the time (5,lO). In order to clarify this point, PR64FS was adsorbed to pili determined by
J53 (R144drd3) for 3 h (seeabove), the phage being identified using specific antibody labeling. Figure 5 clearly shows an S-shaped PR64FS virion (double arrows) attached to the tip of a thin I, pilus (no adsorbed antibodies) protruding from an aggregate, the phage-to-pilus junction being at the top. A short thick I1 pilus is also present (single arrow), distinguishing the two pilus types. At least 20 examples of PR64FS virions adsorbed to thin I pili were noted on an electron microscope support grid. Obviously it would be difficult to demonstrate experimentally that phage PR64FS definitely did not adsorb to thick I, pili as well; however, no such attachment was detected. An RNA-containing bacterial virus called phageIa adsorbsto the sides of thin I, pili (6) and the same comment applies to it. Phage ICXalmost certainly does not adsorb to thick II pili because RNA-containing phagesare highly specific and only attach to one kind of pilus (see Ref. (6)). Bacteria carrying derepressedIncIz plasmids are lysed by lipid phage PR4, while those carrying derepressed IncI, plasmids are not. There are thus no bacteriophagesspecific for thick I, pili available at the moment. As with thick I2 pili, the discovery of thick I, pili raises a number of questions, the most important concerning their possible involvement in conjugation. Studies are continuing in an attempt to answer this, and a search for thick I, pili determined by other plasmids, particularly repressed ones, is in progress. Thick B and K pili are also being sought.
ACKNOWLEDGMENTS I thank Jeannette Fleming for expert technical assistance, and the Medical Research Council of Canada for financial support (Grant MA5608).
FIG. 3. The same plate as in Fig. 1; this thick I, pilus shows the pointed tip clearly. Bar = 100 nm. FIG. 4. A thick I, pilus from J53(R64drdll) Bar = 100 nm.
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lying beside a thin Ii or perhaps a Type I pilus.
FIG. 5. A PR64PS phage virion (double arrows, labeled with antibodies) adsorbed to the tip of a thin I, pilus (no antibodies) protruding from an aggregate. A short thick Ii pilus has a single arrow. Bar = 100 nm.
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REFERENCES 1. BRADLEY, D. E., J. Gen. Microbial.
102, 349-363
(1977). 2. BRADLEY, D. E., Plasmid 4, 155-169 (1980). 3. BRADLEY, D. E., AND COETZEE, J. N., J. Gen. Microbiol. 128, 1923-1926 (1982). 4. BRADLEY, D. E., AND MEYNELL, E., J. Gen. Microbiol. 108, 141-149 (1978). 5. COETZEE, J. N., SIRGEL, F. A., AND LECATSAS, G., J. Gen. Microbial. 117, 547-55 I (1980). 6. COETZEE, J. N., BRADLEY, D. E., AND HEDGES, R. W., J. Gen. Microbial. 128,2797-2804 (1982). 7. LAWN, A. M., Nature (London) 214, 115 1-I 152 (1967).
8. LAWN, A. M., AND MEYNELL, E., J. Hyg. (Cambridge) 68, 683-694 (1970). 9. MEYNELL, G. G., AND LAWN, A. M., Nature (London) 217, 1184-1186 (1968). 10. MEYNELL, E., MEYNELL, G. G., AND DATTA, N., Bacterial. Rev. 32, 55-83 (1968). 11. SHAPIRO, J. A., In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 601-704. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1977. 12. STANISICH, V. A., J. Gen. Microbial. ( 1974).
84, 332-342
9, 331-334 (1983)
Derepressed Plasmids of Incompatibility Group I, Determine Two Different Morphological Forms of Pilus DAVID E. BRADLEY Faculty of Medicine. Memorial University of Newfoundland, St. JohnS, Newfoundland AlB 3V6, Canada Received November 2, 1982 IncI, plasmids R144drd3 and R64drdll determined thick, probably rigid pili in addition to the well-known thinner I, pili. Electron microscopy revealed that the thick I, pili often had large knobs at one end and were pointed at the other. They did not appear to act as receptors for any I-specific bacteriophages.
Bacterial drug resistance plasmids specifying sensitivity to the filamentous bacteriophage If1 (9) were originally classified into severalincompatibility groups forming the socalled I complex (for a listing, see Ref. (2)). However, they have since been tentatively rearranged into only two groups, IncI, and IncI, (6). Receptors for phage If1 and the similar isolate PR64FS (5) are believed to be the serologically distinct thin flexible pili determined by IncI, and IncIz plasmids (5,10), called thin I1 and thin I2 pili, respectively. IncB and IncK plasmids also determine morphologically similar thin flexible pili (2) which are probably not If1 receptors since theseplasmids do not specify sensitivity to If 1. Recently Bradley and Coetzee(3) found that IncIz plasmids determined thick pili as well (thick 12 pili). They were morphologically distinct from thin IZ pili. They acted as receptors for the lipid-containing phage PR4 (12) but not for If1 or PR64FS. In this communication the results of a successfulsearch for thick pili determined by IncI, plasmids (thick I, pili) are described. Escherichia coli 553 (pro met) was used as plasmid host. The IncI, plasmids were R 144drd3 (specifies kanamycin resistance) and R64drdll (specifies streptomycin and tetracycline resistances), both being kindly supplied by N. Datta (see Ref. (II)). Phage PR64FS was kindly donated by J. N. Coetzee.
Brain heart infusion agar (BBL) was used for bacterial growth at 37°C throughout. Drug concentrations were kanamycin, 100 &ml, and tetracycline, 25 clg/ml. Pili were mounted for electron microscopy as described before (2), bacteria being incubated overnight on plates containing appropriate drugs. Phage plus pilus mixtures were prepared as previously described (3). Phage PR64FS was labeled with antibodies for immune electron microscopy by the grid-labeling method (7). Antiserum was raised in rabbits by standard methods (kindly supplied by J. N. Coetzee).All specimens were negatively stained with a mixture of equal volumes of 2% sodium phosphotungstate and 0.1 M ammonium acetate solutions. The discovery that IncIz plasmids determined two distinct morphological types of pilus, thin I2 and thick I2 pili, suggestedthat a similar situation might exist for IncI, plasmids. Two transfer-derepressed IncI, plasmids have been found to determine thick rods. Thesewere morphologically distinct from thin I, pili and appeared different from thick Iz pili in the electron miscoscope. Strain J53(R144drd3) synthesized very many rods with large terminal knobs (Fig. 1). They were approximately 10.5 nm thick (depending on negative staining conditions) compared with the 6 nm of thin I, pili which formed aggregates (Fig. 2). A few Type I pili (7 nm thick)
331
0147-619X/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All ngbts of reprcduction in any form reserved
332
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FIG. 1. Thick I, pilus (longer than average) from an overnight plate of J53(Rl44&3).
Bar = 100 nm.
FIG. 2. The same plate used in Fig. 1, showing a thick I, pilus with a broken knob, and parts of thin 1, pilus aggregates.Bar = 100 nm.
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were present, but these did not aggregate(not illustrated). The knob on the pilus in Fig. 2 seemsto be disintegrating and its appearance suggeststhat it is some form of membranous structure similar to those found on F pili (8), F0 lac pili (4), and D pili (I). Not all thick 11 pili had knobs, which were believed to be located at the proximal end of the pilus like the similar structures just mentioned. The distal tips of the rods were pointed (best seenin Fig. 3). The IncIr plasmid R64drdll also determined thick pili with knobs, but in smaller numbers. Figure 4 illustrates a knob smaller than those shown for R144drd3, but there was considerable size variation with both plasmids. A probable thin I, pilus, or perhaps a Type I pilus, is lying beside the thick one, emphasizing the large difference in thickness. While the thick pili with knobs superficially resemble tailed bacteriophages, they are not bacteriophages since phages have heads of uniform size and tails of constant length. The appearance of the thick pili of both plasmids was consistent with a rigid form (2). Using identical specimen preparation methods, no thick pili could be found for strain 553 without a plasmid (continuous search in the electron microscope for 15 min). Improved electron microscope specimen preparation techniques are undoubtedly the reason why thick II and I2 pili have only been found recently. The distinguishing feature of I complex plasmids is that they support the multiplication of the serologically related filamentous phages If1 and PR64FS. Both adsorb to the tips of conjugative I pili, but none of the published electron micrographs reliably indicates whether these are thick or thin I, or I2 pili, which had not, of course, been distinguished at the time (5,lO). In order to clarify this point, PR64FS was adsorbed to pili determined by
J53 (R144drd3) for 3 h (seeabove), the phage being identified using specific antibody labeling. Figure 5 clearly shows an S-shaped PR64FS virion (double arrows) attached to the tip of a thin I, pilus (no adsorbed antibodies) protruding from an aggregate, the phage-to-pilus junction being at the top. A short thick I1 pilus is also present (single arrow), distinguishing the two pilus types. At least 20 examples of PR64FS virions adsorbed to thin I pili were noted on an electron microscope support grid. Obviously it would be difficult to demonstrate experimentally that phage PR64FS definitely did not adsorb to thick I, pili as well; however, no such attachment was detected. An RNA-containing bacterial virus called phageIa adsorbsto the sides of thin I, pili (6) and the same comment applies to it. Phage ICXalmost certainly does not adsorb to thick II pili because RNA-containing phagesare highly specific and only attach to one kind of pilus (see Ref. (6)). Bacteria carrying derepressedIncIz plasmids are lysed by lipid phage PR4, while those carrying derepressed IncI, plasmids are not. There are thus no bacteriophagesspecific for thick I, pili available at the moment. As with thick I2 pili, the discovery of thick I, pili raises a number of questions, the most important concerning their possible involvement in conjugation. Studies are continuing in an attempt to answer this, and a search for thick I, pili determined by other plasmids, particularly repressed ones, is in progress. Thick B and K pili are also being sought.
ACKNOWLEDGMENTS I thank Jeannette Fleming for expert technical assistance, and the Medical Research Council of Canada for financial support (Grant MA5608).
FIG. 3. The same plate as in Fig. 1; this thick I, pilus shows the pointed tip clearly. Bar = 100 nm. FIG. 4. A thick I, pilus from J53(R64drdll) Bar = 100 nm.
333
lying beside a thin Ii or perhaps a Type I pilus.
FIG. 5. A PR64PS phage virion (double arrows, labeled with antibodies) adsorbed to the tip of a thin I, pilus (no antibodies) protruding from an aggregate. A short thick Ii pilus has a single arrow. Bar = 100 nm.
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COMMUNICATIONS
REFERENCES 1. BRADLEY, D. E., J. Gen. Microbial.
102, 349-363
(1977). 2. BRADLEY, D. E., Plasmid 4, 155-169 (1980). 3. BRADLEY, D. E., AND COETZEE, J. N., J. Gen. Microbiol. 128, 1923-1926 (1982). 4. BRADLEY, D. E., AND MEYNELL, E., J. Gen. Microbiol. 108, 141-149 (1978). 5. COETZEE, J. N., SIRGEL, F. A., AND LECATSAS, G., J. Gen. Microbial. 117, 547-55 I (1980). 6. COETZEE, J. N., BRADLEY, D. E., AND HEDGES, R. W., J. Gen. Microbial. 128,2797-2804 (1982). 7. LAWN, A. M., Nature (London) 214, 115 1-I 152 (1967).
8. LAWN, A. M., AND MEYNELL, E., J. Hyg. (Cambridge) 68, 683-694 (1970). 9. MEYNELL, G. G., AND LAWN, A. M., Nature (London) 217, 1184-1186 (1968). 10. MEYNELL, E., MEYNELL, G. G., AND DATTA, N., Bacterial. Rev. 32, 55-83 (1968). 11. SHAPIRO, J. A., In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 601-704. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1977. 12. STANISICH, V. A., J. Gen. Microbial. ( 1974).
84, 332-342