Vol. 141, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1980, p. 828-8370021-9193/80/02-0828/10$02.00/0

Determination of Pili by Conjugative Bacterial DrugResistance Plasmids of Incompatibility Groups B, C, H, J, K,

M, V, and XDAVID E. BRADLEY

Faculty ofMedicine, Memorial University ofNewfoundland, St. John's, Newfoundland, Canada AIB 3V6

Representative plasmids from incompatibility groups B, C, H, J, K, M, V, andX were transferred to "bald" strains of Escherichia coli or Salmonella typhi-murium. By using a new technique, pili were detected by electron microscopy foreach incompatibility group. Morphology varied but was similar for plasmidswithin a group. These findings suggest that all conjugative plasmids in theEnterobacteriaceae may determine pili.

Drug resistance plasmids have been classifiedby their inability to coexist in the same bacterialstrain (termed incompatibility). It has beenfound that specific pili are determined by plas-mids of the following incompatibility (Inc)groups: D (2; N. Datta, Plasmids of Medical,Commercial, and Environmental Importance,in press); the four F incompatibility groups (7,20); the I incompatibility groups (15, 20, 22); N(D. E. Bradley, Plasmid, in press); P (1, 23); T(C.-M. To, A. To, and C. Brinton, Abstr. Annu.Meet. Am. Soc. Microbiol. 1975, S276, p. 259);and W (5). In addition, pili have been identifiedfor the single plasmid F.lac (6, 7) and the unas-signed plasmid R485 (3). These pilus types ex-hibit several different morphological forms. Theobject of this work has been to ascertain whetheror not representative plasmids of the remainingincompatibility groups (in Escherichia coli K-12) determine pili, and to show the morpholog-ical forms they may take.To detect pili, plasmids must be transferred to

"bald" strains of bacteria (with no other type ofpilus, and preferably with no flagella). Evenwhen this has been done, conjugative pili areoften very difficult to find, since many plasmidsare naturally repressed for transfer and deter-mine very few pili indeed (21). New experimentalapproaches have been used to surmount thisproblem (see Materials and Methods), and as aresult, pili have been detected for plasmids rep-resenting all those remaining incompatibilitygroups: B, C, H, J, K, M, V, and X.

Characterization has been limited to the gen-eral morphology of the pili to demonstrate thedifferent forms encountered. Quantitative lengthmeasurements of detached pili are not consid-ered reliable since pili often break, but where apilus type is exceptionally long it is noted. Someshort pili are obviously unbroken (detectable by

both clear basal structure and defined terminalmorphology), and this too is noted. Pilus thick-ness, an important character, is difficult to meas-ure accurately. Fortunately, thickness differ-ences are very obvious to the naked eye andbear out the approximate measurements pro-vided here. A third important characteristic isflexibility. Some pili such as those of IncN plas-mids are straight, rigid, and easily broken (D. E.Bradley, in press), whereas others, like F pili,are flexible and bend through large angles with-out "snapping". Whether or not a pilus formsaggregates in suspension reflects a property ofthe pilin (pilus protein). This has been notedwhen obvious.To avoid confusion, pili have been named by

the letter of their incompatibility group, a prac-tice now universally adopted.

MATERIALS AND METHODSBacterial strains and plasmids. Table 1 lists the

plasmids used. They were all supplied by N. Datta orR. W. Hedges, save for TEMdrd, which was given byR. J. Pinney. Other sources listed in Table 1 are thosefrom whom the plasmids first originated. They wereall supplied in E. coli K-12 (strains J53 or J62). Theywere transferred by standard mating procedures tomutants resistant to nalidixic acid, streptomycin, orrifampin, of the nonpiliated strains E. coli JE2571 (leuthr fla pil str), E. coli CR34 (leu thr thi lacY thy pil),or Salmonella typhimurium LT2 strain SQ1139 (purCproA ilv str fla pil) being supplied by R. Iyer, R. H.Olsen, and R. Bradley, respectively.Media. BBL brain heart infusion broth or agar and

BBL Mueller Hinton agar (where sulfonamide resist-ance was indicated) were used throughout. The con-centrations of drugs used for plasmid selection were:ampicillin, 400 ,ug/ml; chloramphenicol, 25 ,ug/ml; kan-amycin, 100 jig/ml; sulfonamide, 1 mg/ml; and, tetra-cycline, 10 ,g/mI. Concentrations used for counterse-lection against donors were: nalidixic acid, 20 iLg/ml;rifampin, 100 ,g/ml; and streptomycin, 200 jg/ml.

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Incubation was normally at 37°C, but 270C was usedfor the growth of strains carrying temperature-sensi-tive plasmids (IncH).

Electron microscopy. To detect the very smallnumbers of pili expected to be determined by plasmidsnaturally repressed for transfer, the following methodswere used. In the first, an overnight streak culture ofa nonpiliated strain carrying the plasmids was pre-pared. A large amount of bacteria (a knob measuringvery approximately 2 by 2 by 1 mm) was removed ona 3-mm loop, which was then dipped in a drop ofneutral 0.1 M ammonium acetate solution. The cellswere completely suspended in the small amount ofliquid remaining on the loop on a plastic petri dish.Next, a carbon-coated electron microscope specimensupport grid was touched onto the surface of this verythick suspension. The cells were then almost com-pletely removed from the grid by dabbing it manytimes on the surface of two different dishes of ammo-nium acetate solution (without wetting the upper sur-face of the grid), the second being used when the firstbecame dirty. When visual inspection under a lampshowed that most of the bacteria had been removed(a very faint cloudiness was allowed to remain if pos-sible), the grid was floated for about 1 min on fresh 0.1M ammonium acetate solution. It was finally nega-tively stained with a mixture of equal amounts of 0.1M ammonium acetate and neutral 2% sodium phos-photungstate solution. This successful method wasthought to produce, in effect, a concentrated pilussuspension, many pili becoming detached from cells

TABLE 1. Plasmids

Plasmid Inc Relevant Source/reference'group markers SR 16 B Ap Sm Su 9R40a C Ap Km Su Y. A. Chabbert, 26RAI C Tc Su 14pHH1343a C Ap Sm Tc Su N. DattaR27 HI Tc 11R478 H2 CmKmTc 16R391 J Km J. N. Coetzee, 26R997 J Ap SmSu 19R387 K Sm Cm 14pTM559 K Km Tp C. Monti-BragadinpIE316 K Tc H. TschapecR446b M Sm Tc R. W. Hedges, 26R471a M Ap KmCm 16R753 V Ap Sm Cm Su 13R6K X Ap Sm 18TEMdrd X Ap Sm R. J. PinneydTP227 X Ap J. Frost, H. R.

Smith"TP228 X Km Sm Tc Su J. Frost, H. R.

Smith'TP231 X Ap Cm Tc J. Frost, H. R.

Smith'R485 Su R. W. Hedges, 26

aAp, Ampicillin; Cm, chloramphenicol; Km, kanamycin;Sm, streptomycin; Su, sulfonamides; Tc, tetracycline.

'In some cases reference 26 is cited for a complete bibli-ography.

cWermgerode, East Germany.d School of Pharmacy, University of London, England.e Enteric Reference Laboratory, Colindale Ave., London,

England.

when the large amount of bacteria was suspended inthe small volume ofammonium acetate solution. Thus,areas away from cells were scanned for pili in theelectron microscope, although pili protruding fromcells were also sought. Because of this qualitativeapproach, and because pili were sheared from cells,the numbers of pili per cell could not be estimated.The second method made use of the fact that some

plasmids are temporarily derepressed for transfer im-mediately after being transferred to a new host (21),so that many pili will be produced for a time. Whatwas effectively an isogenic plate mating was carriedout. The donor (E. coli JE2571 carrying a plasmid)and recipient (JE2571 resistant to a counterselectingdrug other than nalidixic acid, a mating inhibitor) werestreaked from overnight selective plates onto freshones by using a heavy inoculum and incubated forabout 6 h. A single drop of broth was then placed onthe center of a transconjugant-selecting plate. Loop-fuls of the donor and recipient cultures, as nearly thesame size as possible (very approximately 1 mm3) werethen suspended in the drop, which was well mixed.The resulting suspension was spread over the plate byusing a glass spreader. After incubation overnight,confluent growth appeared, and bacteria from thiswere mounted as described for the previous method.This is referred to as the temporary derepressionmethod, which was used when few or no pili werefound by the previous method. Except in cases wherepili were numerous, at least two preparations werenormally examined so that a minimum of 10 to 20 pilicould be studied, although not all were photographed.Whether or not pili aggregated significantly could

be assessed in the electron microscope from prepara-tions using derepressed plasmids or those which pro-duced many pili using the temporary derepressionmethod. In these cases, aggregation (or the lack of it)took place in the 0.1 M ammonium acetate solutionused. Since aggregation depends upon several factorssuch as pH, the results apply only to the conditionsmentioned.

Preparation of cell-free pilus suspensions. Incases where no aggregation could be detected in prep-arations made directly from bacteria, a cell-free sus-pension was prepared in M9 salts solution (NH4Cl, 1.0g/liter; KH2PO4, 3.0 g/liter; Na2HPO4, 6.0 g/liter).This was done by using a Thomas Teflon tissue grinderto shear pili from a concentrated bacterial suspension,cells being finally removed by centrifugation (2).

RESULTS

Inevitably some cell debris, which was easilyrecognized as "vesicles," is present in the micro-graphs shown. To facilitate direct comparison,all the illustrations of the different pilus typesare at the same magnification. This compromiseshows a reasonable amount of detail and at thesame time gives an impression of the generalappearance of the pilus. The letter at the bottomof each micrograph indicates the incompatibilitygroup. Due to the limitations of negative stain-ing, it is difficult to measure pilus widths accu-

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830 BRADLEY

rately, so the figures given should be treated asgood estimates rather than accurate measure-ments. Major thickness differences are, however,quite obvious to the eye. Where the numbers ofpili are referred to as being small, only one ortwo could be located on a single grid square(sometimes even fewer).B pili. The representative IncB plasmid, R'6,

determined thin flexible pili (Fig. 1, thicknessabout 6.0 nm) in E. coli strains CR34(R'6) andJE2571(R16). They were consistently producedin much larger numbers by the latter, showingintense aggregation without the requirement ofa cell-free pilus suspension. B pili were muchthinner than F pili (thickness 9.0 nm) and ap-peared to resemble I pili (20), which may besignificant since IncB plasmids show considera-ble DNA homology with those of IncIa (10, 12).C pili. The prototype IncC plasmid is R40a

(8), but no pili could be detected in E. colistrains carrying it by using the temporary de-repression method. However, a second IncCplasmid, RA1, determined numerous pili resem-bling those of F (Fig. 2) in the background strainE. coli JE2571. RAl was originally thought tobe the prototype of an incompatibility group A.This has now been discontinued (N. Datta, inpress), since RAl was later found to be IncC.Very few pili were also determined by the IncCplasmid pHH1343a in strain JE2571, no increasebeing obtained with the temporary derepressionmethod. C pili were about 9.0 nm thick andflexible. IncC plasmids are transferable to Pseu-domonas aeruginosa, in which they are mem-bers of incompatibility group P-3 (17).H1 and H2 pili. Incompatibility group H is

divided into subgroups Hi and H2. IncH1 plas-mids are incompatible with those of IncH2 butshow little DNA homology with them. The plas-mid MIP233, which confers the ability to metab-olize saccharose, may constitute another sub-group IncH3 (25), but has not been includedhere. The representative IncH1 plasmid R27 de-termined straight pili in E. coli JE2571 (Fig. 3).H1 pili were apparently pointed at one end (Fig.4, arrow on right). Observations of a number ofpili suggested that they were rigid, appearing tobreak when bent through a sharp angle (Fig. 4,left arrow). They were about 11.0 nm thick andresembled N pili, which however are only 9.0nm thick (D. E. Bradley, in press). StrainJE2571(R478), which carries an IncH2 plasmid,produced very small numbers of pili apparentlyidentical with those of R27; Fig. 5 was unique,with several H2 pili (arrow points to probablebreak) being found together. Sufficient H1 piliwere present in specimens made directly frombacteria, and in a cell-free suspension from

JE2571(R27) to show aggregation of HI pili, butno observations could be carried out with H2 pilibecause these were insufficient.J pill. The prototype IncJ plasmid, R391,

determined numerous pili in E. coli strains CR34and JE2571 (Fig. 6). They were flexible, about10 nm thick, and very long; one example meas-ured 6.5 Am. They showed no obvious tendencyto aggregate either in suspension or in prepara-tions made directly from bacteria. Another IncJplasmid, R997 (19), determined similar long pili(Fig. 7) in strain JE2571. The numbers presentwere consistent with those of a plasmid dere-pressed for pilus synthesis, and should be visibleusing standard methods for examining cells.K pill. Plasmids of incompatibility group K

determined very few pili unless the temporaryderepression method was used, when very largenumbers were produced; the plasmids testedwere thus naturally repressed. E. coliJE2579(pTM559) produced sufficient pili todemonstrate intense aggregation (Fig. 8). Simi-lar pili were determined in the same backgroundstrain by the prototype plasmid R387 (Fig. 9)and by another IncK plasmid pIE316. K piliwere indistinguishable from B pili in morphology(flexible, 6.0 nm thick) and in their ability toaggregate.M pill. It has recently been found that IncL

plasmids are incompatible with IncM plasmids(24), so that the former group has been discon-tinued. The prototype IncM plasmid, R446b,and the old IncL prototype, R471a, have there-fore been included here. E. coli JE2571(R446b)and JE2571(R471a) both produced a few shortpili (apparently pointed), but very many morewhen the temporary derepression method wasused. As a further check, each plasmid was trans-ferred to S. typhimurium LT2 strain SQ1139,similar pili being synthesized (Fig. 10 and 11). Mpili resembled N pili in that they were short andrigid. They appeared to have a marked basalstructure (Fig. 10, arrow), lacking in N pili. Ob-servations of pili extended from cells (not illus-trated) showed that it was the distal end thatwas pointed. M pili were often very short (Fig.11), resembling small nails.V phli. The IncV plasmid R753 was trans-

ferred to E. coli JE2571 in which it determinedvery few 9.0-nm thick, flexible pili (Fig. 12). Noincrease in numbers could be obtained by usingtemporary derepression, and insufficient piliwere present in a pilus suspension to assessaggregation, although pili were occasionally seensticking together (Fig. 12).X pill. IncX plasmids have been well studied,

particularly the prototype, which is the unusualmulticopy plasmid R6K (18); it apparently de-

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PILI OF CONJUGATIVE PLASMIDS 831

termined no pili (3). R71lb and R778b were alsooriginally classified as IncX, but it has beenfound that, unlike R6K, they determine thickflexible pili resembling those of F and C plas-mids, and that they confer sensitivity to thefilamentous bacteriophage fd (2). Although theyare compatible with plasmids of the F incom-patibility groups, their incompatibility relation-

.:~~ ~ ~.q~r. z & ,

at$Zao,xFd

45

.:-f' ;'>'..s..B ships with other IncX plasmids are not clearlydemonstrable. Being undoubtedly incompatiblewith one another, they have been assigned a newgroup, IncD (see N. Datta, in press). R485 de-termines extremely thin pili, only about 5.0 nmthick (3). Although it is apparently incompatiblewith R6K, its status remains uncertain. Forthese reasons, special attention has been paid to

i .2 K;. ;..

A

FIG. 1. B pili from E. coli JE2571(R'6) prepared from an overnight selective plate. They show a tendencyto aggregate.

FIG. 2. C pilus from E. coli JE2571(RA1) prepared from an overnight selective plate.FIG. 3. Hi pilus from a suspension made by using E. coli JE2571(R27), showing typical straight rigid

appearance. Bar represents 100 nm for all three figures.

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832 BRADLEY

-e 4.>s8fiN -*;4;' V *

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is^; H2-FIG. 4. H1 pili from E. coli JE2571(R27) by the temporary derepression method. Broken pilus, left arrow,

and pointed tip, right.FIG. 5. H2 pili from an overnight selective plate of E. coli JE2571(R478) showing a broken pilus (arrow).

Bar represents 100 nm for both figures.

the group, several plasmids being examined.X pili (Fig. 13) were observed with E. coli

strains CR34(R6K) and JE2571(R6K).JE2571(TEMdrd), which carried a derepressedversion of R6K, produced many more pili thanthe latter, as would be expected. Although no

pili could be found by the direct examination ofstrains carrying three other IncX plasmidsTP227, TP228, and TP231, the temporary de-repression method yielded large numbers of fil-aments, apparently identical with those ob-tained with TEMdrd and R6K. The temporary

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FIG. 6. Jpili from an overnight selective plate of E. coli CR34(R391).FIG. 7. J pilus from an overnight selective plate of E. coli JE2571(R997). Bar represents 100 nm for both

figures.

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i ~~~KK MFIG. 8. Aggregated Kpili from E. coli JE2571(pTM559), using the temporary derepression method.FIG. 9. Single Kpilus from an overnight selective plate of E. coli JE2571(R387).FIG. 10. M pilus from an overnight selective plate of S. typhimurium SQ1139(R446b) showing the basal

structure (arrow) and the distal point (bottom).FIG. 11. Short Mpili from an overnight selective plate of S. typhimurium SQ1139(R471a). The pili have

been partly penetrated by the negative stain (center). Bar represents 100 nm for all four figures.

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PILI OF CONJUGATIVE PLASMIDS 835

..m-.X ..;

FIG. 12. V pili from an overnight selective plate of E. coli JE2571(R753). There was some evidence ofaggregation, though there were too few pili to be certain.

FIG. 13. X pili from E. coli JE2571(TP231) using the temporary derepression method. No pili could befound for this strain using an overnight selective plate. The micrograph does not show that the pili aggregatesignificantly. Bar represents 100 nm for both figures.

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836 BRADLEY

derepression method also increased the numbersof pili produced by strain JE2571(R6K).X pili were about 9.0 nm thick and were

unusually flexible (Fig. 13). They tended to ag-gregate (not evident from Fig. 13). X pili weremorphologically different from D pili (2), whichare straighter, and from pill of R485, which aremuch thinner. Strain JE2571(R485) was checkedfor the presence ofX pili in addition to the thinones, by using the temporary derepressionmethod, but only the thin ones could be found.These results add to the uncertainty of the IncXstatus of R485, which therefore remains unas-signed. The observations described concur withthe placing of R71lb and R778b in the newgroup IncD. It would appear that, except forTEMdrd, the IncX plasmids studied here werenaturally repressed.

DISCUSSIONThese results clearly demonstrate that pili are

determined by conjugative plasmids represent-ing all the incompatibility groups in the Enter-obacteriaceae. The characteristics of the pilldescribed are summarzed for convenient refer-ence in Table 2, which includes the properties ofF and P pili for comparison. Two basic forms ofpilus appear to exist-flexible and rigid-andalthough these occur with varying thicknesses,pili of unique morphology are not found for allincompatibility groups. A characteristic whichreflects differences in the chemical nature ofpilin is whether or not aggregates are formed inpilus suspensions, as is the case with F pili (7).Most of the pilus types described aggregate sig-nificantly. However, the characteristic may be

TABLE 2. Characteristics ofpili

(InC)ua Morphology (Tnm) AggregationdB Flexible 6 +C Flexible 9FI Flexible 9 +Hi Rigid? 11 +H2 Rigid? 11 NTJ Flexible 10K Flexible 6 +M Rigid 11 -P Rigid 8 +V Flexible 9 NTX Flexible 9 +

a Well-studied pili of two other important incom-patibility groups have been included for comparison(FI, P).

b Pilus thicknesses were estimated from negativelystained preparations, and due to inherent inaccuracies,are only given to the nearest nanometer.

e+, Aggregation; -, none detectable; NT, no testdue to insufficient pili.

useful in distinguishing C pili from F pili, forexample. The ability of a pilus to act as a recep-tor for pilus-specific bacteriophages is anotherdistinguishing characteristic, and it will be inter-esting to see if any can be isolated for the plas-mids described here. Perhaps the most reliableway of distinguishing pili is by immune electronmicroscopy in which it is determined whether ornot specific antibodies to one pilus type reactwith (adhere to) pili of another type. An exten-sive serological investigation of pili in all theincompatibility groups is in progress. It appearsso far that identical pili are not determined byplasmids in different incompatibility groups.These results suggest that it is highly unlikely

that a transfer system without pili exists, at leastin the Enterobacteriaceae. For a number ofyears it was thought that plasmids of such im-portant incompatibility groups as N and X didnot determine pili, but this is not the case.Although it is likely that such pili are involvedin conjugation and genetic transfer, this has yetto be demonstrated unequivocally for the major-ity. It has proved to be the case for those systemsso far investigated, which include IncFI (7),IncN, and IncP (D. E. Bradley and T. Chaud-huri, In C. Stuttard and K. Rozee, ed., Plasmidsand Transposons: Environmental Effects andMaintenance Mechanisms, in press) and alsoIncW (4).

It will have been noted that the temporaryderepression method works particularly well inthe cases of IncK, IncM, and IncX plasmids.This clearly indicates that these plasmids arenaturally repressed for pilus synthesis and pre-sumably transfer. In other cases such as IncV,very few pili could be found by any method, andthey too are probably repressed. Although aquantitative approach with regard to the num-ber of pili synthesized may be difficult, it ishoped to correlate piliation and transfer fre-quency (under optimum conditions) in a futurestudy. It seems highly likely that the pili ofsomerepressed plasmids (perhaps the IncC R40a) willnot be detectable in the electron microscopebecause too few are determined. In such cases itwill be necessary to construct derepressed mu-tants to study them.

ACKNOWLEDGMENTSI am particularly grateful to Naomi Datta and Bob Hedges

for providing so many plasmids without which it would nothave been possible to carry out the work. I also thank R. J.Pinney for TEMdrd, those who donated bacterial strains, andNeil Willetts for valuable discussions suggesting the possibilityof using temporary derepression in initial plasmid transfer toobtain more pili. Doris Cohen provided excellent technicalassistance, in particular carrying out the very numerous strainconstructions.

This work was supported by grant MA5608 from the Med-ical Research Council of Canada.

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LITERATURE CMD

1. Bradley, D. E. 1974. Adsorption of bacteriophages spe-cific for Pseudomonas aeruginosa R factors RP1 andR1822. Biochem. Biophys. Res. Commun. 57:893-900.

2. Bradley, D. E. 1977. Characterization of pili determinedby drug resistance plasmids R711b and R778b. J. Gen.Microbiol. 102:349-363.

3. Bradley, D. E. 1978. Determination of very thin pili bythe bacterial drug resistance plasmids R485. Plasmid 1:376-387.

4. Bradley, D. E. 1978. W pili: characteristics and interac-tion with lipid phages specific for N, P-1, and W groupplasmids, p. 339-353. In D. E. Bradley, E. Raizen, P.Fives-Taylor, and J. Ou (ed.), Pili. International Con-ferences on Pili, Washington, D.C.

5. Bradley, D. E., and D. R. Cohen. 1976. Basic character-ization of W-pili. J. Gen. Microbiol. 97:91-103.

6. Bradley, D. E., and E. Meynell. 1978. Serological char-acteristics of pili determined by the plasmids R71lb andF.lac. J. Gen. Microbiol. 104:141-149.

7. Brinton, C. C. 1971. The properties of sex pili, the viralnature of "conjugal" genetic transfer systems, and somepossible approaches to the control of bacterial drugresistance. Crit. Rev. Microbiol. 1:105-160.

8. Chabbert, Y. A., M. R. Scavizzi, J. L. Witchitz, G. R.Gerbaud, and D. H. Bouanchaud. 1972. Incompati-bility groups and the classification of fi- resistancefactors. J. Bacteriol. 112:666-675.

9. Datta, N., and J. Olarte. 1974. R factors in strains ofSalmonella typhi and Shigella dysenteriae isolatedduring epidemics in Mexico: classification and compat-ibility. Antimicrob. Agents Chemother. 5:310-317.

10. Falkow, S., P. Guerry, R. W. Hedges, and N. Datta.1974. Polynucleotide sequence relationships amongplasmids of the I compatibility complex. J. Gen. Micro-biol. 85:65-76.

11. Grindley, N. D. F., J. N. Grindley, and E. S. Anderson.1972. R factor compatibility groups. Mol. Gen. Genet.119:287-297.

12. Grindley, N. D. F., G. 0. Humphreys, and E. S. An-derson. 1973. Molecular studies of R factor compati-bility groups. J. Bacteriol. 115:387-398.

13. Hedges, R. W. 1975. R factors from Proteus mirabilisand P. vulgaris. J. Gen. Microbiol. 87:301-311.

14. Hedges, R. W., and N. Datta. 1971. fli R factors givingchloramphenicol resistance. Nature (London) 234:220-

221.15. Hedges, R. W., and N. Datta. 1973. Plasmids determin-

ing I pili constitute a compatibility complex. J. Gen.Microbiol. 77:19-25.

16. Hedges, R. W., V. Rodriguez-Lemoine, and N. Datta.1975. R factors from Serratia marcescens. J. Gen. Mi-crobiol. 86:88-92.

17. Jacoby, G. A. 1977. Classification of plasmids in Pseu-domonas aeruginosa, p. 119-126. In D. Schlessinger(ed.), Microbiology-1977. American Society for Micro-biology, Washington, D.C.

18. Kontomichalou, P., M. Mitani, and R. C. Clowes. 1970.Circular R-factor molecules controlling pencillinasesynthesis, replicating in Escherichia coli under relaxedor stringent control. J. Bacteriol. 104:34-44.

19. Matthew, M., R. W. Hedges, and J. T. Smith. 1979.Types of fl-lactamase determined by plasmids in gram-negative bacteria. J. Bacteriol. 138:657-662.

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