dec. f+, hfr, f' strains of salmonella abony · s. abony. all strains of s. abony used are...

BACTERIOLOGICAL REVIEWS, Dec. 1972, p. 608-637 Vol. 36, No. 4Copyright i 1972 American Society for Microbiology Printed in U.S.A.

F+, Hfr, and F' Strains of Salmonellatyphimurium and Salmonella abony

KENNETH E. SANDERSON,1 HELEN ROSS, LILJA ZIEGLER, AND P. HELENA MAKELADepartment of Biology, University of Calgary, Calgary, Alberta, Canada, and Central Public Health

Laboratory, Helsinki, Finland

INTRODUCTION .............................................................. 609MATERIALS AND METHODS ...................... ........................... 609Media..................................................................... 609Strains .................................................................... 609

S. typhimurium ............................................................. 609S. abony .................................................................... 612

Mating Methods ............................................................... 6121. Replica plating............................................................ 6132. Plate mating.............................................................. 6133. Broth mating.............................................................. 6134. Interrupted conjugation .............. .................................... 613

Isolation of Hfr Strains ......... ............... .............................. 613Isolation of F' Strains .......................... ............................... 613

1. Selection for early entry of a distal gene ........ ......................... 6132. Selection for recombinants for proximal or distal genes ...... ............ 6133. Selection for "transmissible Hfr property"................................. 613

Transmission of the F Factor ...................... ........................... 613Testing for the Presence of a '4ansmissible F Factor ........ .. ............... 613Storage Methods .............................................................. 614

1. Stab cultures.............................................................. 6142. Slant cultures............................................................ 6143. Frozen cultures............................................................ 6144. Lyophilized cultures .................. .................................... 614

Methods of Testing Phage Sensitivity .................. ....................... 614Nomenclature ................................................................. 614

RESULTS .................................................................... 614Mating Methods ............................................................... 614Stability of Hfr Strains During Storage ................ ...................... 617Sources of the F+ Strains ........................ ............................. 618Hfr Strains of S. typhimurium ............ ................................... 618SR305 (HfrA) ............................................................... 618SR315 (leu-256/FS21) ............................................ 618SU418 (HfrB2) .............................................................. 618SU436 (HfrB3) .............................................................. 620SA458 (HfrKl-l), SA464 (HfrKl-2), SA537 (HfrKI-3), SA538 (HfrKl-4),

SA539 (HfrKl-5), SA639 (HfrKl-6), SA642 (HfrKl-7), and SA967(HfrKl-8) .............................................................. 620

SA486 (EHfrK3).............................................................. 623SA534 (HfrK4).............................................................. 623SA535 (HfrK5)............................................................. 624SA536 (HfrK6), SA949 (HfrK7), and SA977 (HEfrK8) ...... .................. 624SA540 (HfrK2-1), SA640 (HEfrK2-2), and SA653 (HfrK2-3) ...... ............ 624SA654 (HfrK9).............................................................. 624SA722 (HEfrKO).............................................................. 624SA828 (HfrK25).............................................................. 624SA949 (HfrK7).............................................................. 624SA949 (HfrKl4), SA967 (HErK-8), and SA975 (HfrK13) ...... .............. 624SA955 (HfrK20) and SA965 (HfrKl7) ......... .............................. 626SA962 (HfrK16).626SA965 (HfrK7).626SA966 (HfrKl9).............................................................. 626SA967 (HfrKI-8)............................................................ 628SA969 (HfrK5).............................................................. 628

'Temporary address, until 30 June 1973: Max-Planck Institut fur Immunbiologie, D 78 Freiburg i. Br.. Germany.608

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F+, Hfr, AND F' STRAINS OF SALMONELLA

SA970 (HfrKll and SA978 (HfrKl2) ........................................SA975 (HfrKl3), SA977 (HfrK8), SA978 (HfrKl2) ..........................

Hfr Strains of S. abony .......................................................HfrHl ......................................................................HfrH2 (SW1403) ............................................................HfrH3, SW1452 .............................................................HfrH4 (SWI446) ............................................................HfrH5 (SW1462) ...........................................................HfrH7 (SH475) ..............................................................HfrHlO .....................................................................HfrH12 (SH671) ............................................................

F' Factors Carrying Salmonella Genetic Material ..............................S. typhimurium Genetic Material ..............................................S. abony Genetic Material .....................;E. coli F' Factors ..............................................................

DISCUSSION ...................................................................LITERATURE CITED ..........................................................

629629629632632632632632633633633633633633634634635

INTRODUCTIONBacterial conjugation as a method of genetic

analysis was used first in Escherichia coli andled to the discovery of the F factor in E. coliK-12 (9, 13). Hfr strains and F' strains were

isolated in this species and were used exten-sively in genetic analysis; a summary of thesestudies is presented in companion papers (17,38).F factors suitable for genetic analysis were

not recognized in Salmonella by the late 1950's.There were several reasons to try to developmaterials for bacterial conjugation in thesespecies. Firstly, the genes involved in biosyn-thesis of several compounds, e.g., histidine,methionine, and cysteine, had been extensivelystudied by P22-mediated transduction, but thelinkage relationships of different transducingfragments could not be determined by trans-duction. Secondly, studies on the immuno-chemistry of Salmonella surface antigens were

well advanced. Thirdly, the Salmonella group

commonly includes pathogenic organisms inwhich it would be possible to study some

determinants of bacterial virulence.The F factor was transferred from E. coli to

Salmonella species (21, 43), and F+, Hfr, and F'strains are now available primarily in S.typhimurium (31, 43) and in S. abony (20). Itmust be emphasized that the above donorstrains of Salmonella and those discussed inthe present report are not hybrids ofSalmonella and E. coli; they carry F from E.coli, but all the data indicate that they carry no

chromosomal genes from E. coli. Hybrid Hfrstrains also have been derived from crosses ofE. coli Hfr x S. typhimurium F-, with selec-tion for the terminal Hfr gene from E. coli, andisolation of Hfr strains with part of the chromo-some of E. coli as well as F; these strains are

not considered here.It is the purpose of this report to describe in

detail these donor strains which have beenisolated in Salmonella over a period of severalyears. Almost all of the strains which are heredescribed are available from the laboratories ofthe authors.

MATERIALS AND METHODS

Media. Modified Davis minimal medium(MM) contained: K2HPO4, 10.5 g; KH2PO4,4.5 g; MgSO,, 0.05 g; (NH4)2SO4, 1.0 g;glucose, 2.0 g; distilled water, 1 liter; this wassolidified where desired by 1.5% agar (Difco).In MM supplemented with L-amino acids,purines, or pyrimidines, the concentration ofsupplement was 20 ,g/ml; for vitamins thesupplement was 2 ,tg/ml. In some tests othersugars, such as xylose or maltose, were used assole carbon source at 2 g/liter. Cells were grownroutinely in Difco nutrient broth plus 20 ,g ofL-cysteine per ml. Streptomycin was used,when indicated, in the concentration of 1mg/ml. Conjugation was carried out in DifcoPenassay broth. The nutrient medium usedroutinely was NA (Difco Penassay broth plus1.5% Difco Bacto-agar). Bile salts sensitivitywas tested with 0.4% (w/v) sodium deoxycho-late in NA. Single enriched (SE) and doubleenriched (DE) media were MM to which hadbeen added 1/80 and 1/40 volumes, respec-tively, of Difco nutrient broth. Fermentation ofsugars was tested on Difco MacConkey agarplus 2% sugar. Saline soft agar was 0.75% agarin an aqueous solution containing 0.85% NaCl.

StrainsS. typhimurium. The parents of most of the

S. typhimurium strains used came from thestock collection of the late M. Demerec and arenow maintained in the Salmonella GeneticStock Centre (SGSC), University of Calgary.The F+ and Hfr strains used are listed in Table1; a few were isolated by N. Zinder, the

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611VOL. 36, 1972 F+, Hfr, AND F' STRAINS OF SALMONELLA


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remainder by K. E. Sanderson and his col-leagues at Brookhaven National Laboratory,New York, or at the University of Calgary. Allthe multiple mutant F- strains of S.typhimurium used in the study are described inTable 2; a number of single auxotroph mutantswhich were used are not listed; these strains aremaintained in the SGSC.

S. abony. All strains of S. a bony used arederivatives of S. abony Ky 103 (7). They wereisolated and maintained by P. H. Makelai andher colleagues at the University of Helsinki orat the Central Public Health Laboratory, Hel-sinki. This S. abony line contains a prophage(PKylO3) that is an A-type phage, whichconverts to the presence of antigen 1, is serolog-

ically related but not identical with P22, andnot coimmune with it (1). Its attachment site isclose to purE at min 19 and is probably sharedby P27 (Makela and Bagdian, submitted forpublication). When present in the donor andnot in the recipient, it can lead to zygoticinduction and loss of a class of recombinants.Derivatives of HfrH1 that were cured of thisprophage were prepared to avoid this, as theprophage was injected early by this Hfr.

Mating MethodsFour different methods were used, shown

here in increasing order of precision. Normally,before proceeding with method 3 or 4, an Hfrstrain was single-colony isolated by method 1

TABLE 2. Recipient strains used in this study

Strain Source Genotype

S. typhimuriumSL680SB93 (= SU674)DB76 (= SA787)

DB99 (= SA1361)SU208SU280SU453SU633SA508SA570

SA571SA572SA624SA1134SA1145SA1234SA1475

SA1476SA1504SA1515SA1601SA1602

SH4905S. abony

SH67SH538SH549SH628SH632

SW1355SW1361SW1373SW1375SW1409SW1414

B. StockerP. HartmnanD. Berkowitz

J. GotsDemerecSGSCSGSCP. HartmanSC SCSGSC

SGSCSGSCN. KredichSU453SC SCSGSCSC SC

SGSCSGSCSC SCDemerecSC SC

Makela

MiikelaMikelaMikeliiMakeliiMiikelii

MakelaMiikeliiMikeliiMikeliMiikeliiMiikelii

_______________ .1 ____________J~~~~~~~~~~~~~~~~~~~~~~~~~~~

purC7proA46 M10fla ilvA405 str-rhisE34 metG319 purG302 str-rpurC7 proA46 ilvA405 rha-461 iMl0 fla-56 fim- mtlA 116 xyl-172

str-rcya purC7 purI590 proA46 ilvA405 rha-461 fla-56 metE strAtrpA8 proA26 cysB12purC243 tyr-41 argEl16hisF1009 trpB2 metA22 xyl-1 strA201metE338 ilvA401 str-rmet-483 cysE396metA22 trpB2 hisF1009 xyl-1 ilvA99 cysE1363 mtl-105rfa-3060 strA201metA22 trpB2 hisFlOO9 xyl-1 ilvA99pyrE231 malAllO strA201metA22 trpB2 hisF1009 xyl-1 ilvA99 pyrE231 malBI1 I strA201cysEl 709pyrE125 xyl-3phe-47 metA22 trpB2 hisF1009 xyl-1 strA201proAB391 metA22 trpB2 hisF1009 xyl-1 ilvA99 strA201argFl18 thi-36metA22 trpB2 hisF009 xyl-1 ilvA99 pyrE231 malAl10 gal-851

strA201purE68 pro-47str-229metE404 purD55 strA201 (P22L4) +

purA58 ara-9argF118 thi-36metA22 trpB2 hisFi009 xyl-1 ilvA99 cysE1363 rfa3060pro-391 strA201trp-294 pyrB92 thr-115 str-573

his-5801 gal-655 Hl -i H2-1, 2 str-505his-5801 argE752 ilvA 102 gal-655 Hl -i H2-1,2 str-505pro-1001 ara-151 gal-656 mal-151 xyl-202 Hl -i H2-1, 2 str-501thr-902 ara-151 gal-656 mal-151 HI -i H2-1,2 str-501arg-751 ara-151 gal-656 mal-151 mtl-203 xyl-203 Hl-i H2-1,2

str-501pro-1001 str-501met-1151 str-501leu-1351serB1202arg-751 str-501pro-1001 leu-1352str-501

612 BACTERIOL. REV.


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and checked by method 2.1. Replica plating. Isolated colonies of the

donor strain were replica plated onto MM orSE medium previously spread with cells of S.typhimurium purC7 F-, pyrB96 F-, or cysE396F-, three strains which give little cross-feeding.S. abony F- strains are preferable with S.abony donors because of higher frequencies ofrecombinants in homologous combinations.MM with streptomycin can be used if therecipient is streptomycin resistant, but thenumber of recombinants will be reduced wherestreptomycin-sensitive donors are used. Thenumber of prototrophic recombinants followingincubation was a measure of the fertility of thedonor strain.

2. Plate mating. A drop of a broth culture ofthe donor strain was placed onto plates of MMspread with cells from broth cultures of auxo-trophic F- strains.

3. Broth mating. A 1-ml amount of a loga-rithmic-phase broth culture of the donor strainwas mixed with 2 ml of an overnight stationary-phase culture of the F- in 7 ml of Penassaybroth and then incubated at 37 C for 60 min.An 0.1-ml amount of the mating mixture and0.1 ml of a 16- 2 dilution of the mating mixturewere plated in a saline-soft agar overlay ontoplates of selective medium which contained,when the recipient was streptomycin resistant,600 gg of streptomycin sulfate per ml. Thenumber of donor cells was determined byplating dilutions of the donor culture, at zerotime, onto NA.

4. Interrupted conjugation. Separation ofmated pairs was accomplished in earlierstudies by a Fisher Mini-Mixer. With recentlyisolated Hfr strains, not all mated pairs wereseparated, so that samples were blended for 20sec in a Micro-jar of a Waring blender.

Isolation of Hfr StrainsNA plates with isolated colonies of an auxo-

trophic F+ strain were velvet-recplicated ontoSE plates spread with cells of one of the F-strains of S. typhimurium purC7, pyrB96, orcysE396. Colonies yielding many recombinantswere picked from the master plate, streaked,and retested. Hfr strains were infrequentlydetected in F+ cultures which were recentlysingle-colony isolated but frequently werefound after storage of the culture on a slant atroom temperature for several months. The sibselection method (3) was used for isolation ofHfrH4, 5, 7, 10, and 12 of S. abony. Approxi-mately 100 cells of the F+ culture were placedin each of 100 test tubes and grown to station-

ary phase. Cells from each tube were tested forrecombination by plate mating methods, andthose tubes giving high numbers of recombi-nants were examined for Hfr strains by replicaplate methods (20).

Isolation of F' Strains1. Selection for early entry of a distal

gene. Recombinants were selected for a genetransferred near the distal extremity of the Hfrchromosome, following interruption of matingafter a short mating period, usually of 30 to 60min (11, 32, 39).

2. Selection for recombinants for prox-imal or distal genes. This type of selection wasmade in crosses of Hfr strains to recipientstrains carrying a recA mutation (16).

3. Selection for "transmissible Hfrproperty." Selection for "transmissible Hfrproperty" (24) is based on the high transmis-sibility of a nonintegrated F factor. The Hfrstrain is grown in mixed culture with a recipi-ent strain, the mixture is retransferred andregrown several times, and then the originalrecipient strain is isolated and tested for donorability by using replica plating or plate matingmethods. If frequent chromosome mobilizationfrom a specific region is detected, the F factorcan be tested to see if it carries genetic materialfor known genes in this region.

Transmission of the F FactorAn F+ strain and an F- strain with distin-

guishable auxotrophy were grown in mixedculture in broth, and single colonies of therecipient type were isolated from the mixture.These colonies were tested for ability to donatetheir chromosome in plate mating and forsensitivity to the male-specific phages MS2and M13.

Testing for the Presence of aTransmissible F Factor

The indicator system includes an E. coli K12F- strain carrying a sex-factor affinity locus,sfa-3; if this strain acquires an F, the Fintegrates at the sfa-3 locus, and the strainbecomes an Hfr of type Hfr3 which injectsO-metA-thr--- (27). This Hfr behavior isshown by transfer of metA + to a second E. coliK12 MetA- F- strain. Equal amounts of astationary-phase broth culture of the twostrains of E. coli K12, W3876 (sfa-3 lac str-r)and W3637 (metA str-r), were mixed andspread on a minimal-lactose-streptomycinplate (24). The strains to be tested for trans-mission of F were applied to the dried plate as

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drops or by replica plating. Transfer of F isindicated by growth of MetA+ Lac+ Str-rcolonies.

Storage MethodsFor donor strains, a high fertility colony was

selected, inoculated to slants, and grown over-night to provide cells for the following fourstorage methods.

1. Stab cultures. Small vials (0.25 dram)containing nutrient-soft agar (0.75%) werestabbed, corked, dipped in liquid paraffin toseal, and stored at room temperature.

2. Slant cultures. Slants containing Difconutrient agar were inoculated, closed with ascrew cap, grown at 37 C, and stored at roomtemperature.

3. Frozen cultures. Cells from a slant weresuspended in sterile saline, centrifuged, sus-pended in 15% glycerol in aqueous saline solu-tion in screw-cap tubes, and stored at -76 C ina low-temperature freezer.

4. Lyophilized cultures. A dense suspen-sion of cells in an aqueous solution containing5% glucose and 5% peptone (Difco) was spottedonto filter-paper strips in lyophil ampoules.These ampoules were evacuated to a pressureof 30 to 60 g of Hg, held for 30 min, sealedunder vacuum, and stored at room tempera-ture. Vaccum was confirmed with a Tesla coilvacuum tester.

Methods of Testing Phage SensitivityA stationary-phase culture of a bacterial

strain was flooded onto a plate of NA anddried, and a drop of each phage, at a titer of ca.108 plaque-forming units per ml, was spotted ontop. The plates were incubated and observedfor lysis (42). Incubation temperature for testswith MS2 (4) was at 42 C rather than 37 C.Phages for determination of the rough orsmooth phenotype were provided by B. A. D.Stocker, of Stanford University; in some casesthe tests were done in his laboratory.MS2 and M13 (10) gave visible lysis of most

F+ and Hfr strains of S. typhimurium, but afew, e.g., SR297 and SR305, did not showvisible lysis. However, these strains containcells which are sensitive to these phages, forthey multiply the phages to high titer in liquidmedium (Sanderson an .d Hall, unpublisheddata). These strains fail to show visible lysisbecause only 10% or less of the cells form visiblesex pili, as seen under the electron microscopeafter labeling with MS2 phage (Sanderson andLawn, unpublished data).

NomenclatureThe genetic nomenclature follows the usage

of Demerec et al. (6). The abbreviations forgene loci, e.g., his for histidine, are listed inother papers (29, 30). In Hfr strains, the strainnumber refers to a particular strain, with aspecific genotype; for example, SR305 has thefollowing genotype: hisD23 gal-50 HfrA. Thedesignation "HfrA" refers to the Hfr proper-ties of the strain; in HfrA, the F factor is in-serted between ilv and pyrE. If this Hfr prop-erty is transferred to a new strain by terminalmarker selection, this new strain will also beHfrA, but with a new strain number. In addi-tion to designations of gene loci, the followingabbreviations are used: LPS, lipopolysaccha-ride; PFU, plaque-forming unit; SGSC, Sal-monella Genetic Stock Centre.

RESULTS

Mating MethodsPreliminary experiments (31, 43) suggested

that in S. typhimurium the frequency of mat-ing in liquid medium is low; this frequencyappeared to be increased by mixing the donorand recipient on a membrane filter and thenincubating the filter on a plate of nutrient agar(25). All the interrupted conjugation data usedin constructing a linkage map of S.typhimurium (31) were obtained in this way.More recently, we found that the frequency ofrecombination from mating on membrane fil-ters is usually no higher than from mating inbroth. The following experiments, intended toreveal the frequency of recombination fromdifferent mating methods, were done with twoHfr strains, SR305 and SA536, and one F-strain, SA570, all of S. typhimurium. SA570has the "rough-sensitive" pattern of phagesensitivity (see Table 3), due to a rough muta-tion, rfa-3060, which maps in the rfa cluster ofgenes at 116 min on the linkage map. Inaddition, SA536 has a Rfa (rough A) pheno-type, based on its phage sensitivity, of ananomalous type (35). The change from rough tosmooth phenotype affects the frequency ofconjugation with a recipient (40, 41), but testswith normal, smooth strains suggest to us thatour conclusions about mating methods areunaffected by the rough phenotype of the abovestrains.The donor and recipient strains were grown

to stationary phase in nutrient broth in un-shaken culture; the donor was diluted 1:20 innutrient broth and grown for 2 hr in unshakenculture to mid-log phase; both donor and

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recipient were then centrifuged and resus-pended in fresh Penassay broth to a turbidity of30 units for the donor and 150 units for therecipient, as measured on a Klett-Summersoncolorimeter with a no. 66 (red) filter. For eachmating, 1.0 ml of the donor and 2.0 ml of therecipient were used; after 60 min of mating, thecells were plated in saline-soft agar on selectiveMM with 600 ug of streptomycin sulfate perml. Alternate mating methods, which use Pen-assay broth, are as follows:

(1) Mix donor and recipient in a 16-ml tube("contact mixture"); then at 5 min transfer theentire contents of the tube to 7 ml of broth in a250-ml flask ("mating mixture").

(2) Add donor and recipient directly to 7 mlof broth in a 250-ml flask.

(3) Mix donor and recipient in a 16-ml tube("contact mixture"); at 5 min, transfer to 97 mlof broth in a 1-liter flask.

(4) Add donor and recipient directly to 97 mlof broth in a 1-liter flask.

(5) Mix donor and recipient on a membranefilter (Millipore), place on nutrient soft agar for5 min, and then transfer filter to 10 ml of brothin a 250-ml flask.

(6) Mix donor and recipient on a membranefilter, place on nutrient soft agar for 5 min, andthen transfer filter to 100 ml of broth in a250-ml flask.Both SR305 and SA536 transfer Ilv+ and

MetA+ as proximal genes, though their pointsof origin are not identical (Table 1; Fig. 1).Method 2 is a broth mating method, similar tothe one used in most of the studies in thisreport, whereas method 5 is a minor modifica-tion of the membrane filter method used inearlier studies (31). The data with SR305(Table 4) indicate that the use of a membranefilter in the "contact period" in method 5 gives4.0 x 10-3 Ilv+ recombinants and 2.6 x 10-3MetA+ recombinants per donor cell, whereasthe number of the recombinants with method 2is only one-third to one-half as many. Thebroth mating method used in early tests resem-bled method 4 but yielded only about one-eighth as many recombinants as method 5,which uses a membrane filter. With SA536, anapproximately equal frequency of recombi-nants is obtained with each different method(Table 4). Crosses with other Hfr and F-strains, both of S. typhimurium and S. abonyas well as of other species such as S.montevideo and S. senftenberg, have also in-dicated that method 2 normally yields as manyrecombinants as method 5; hence, because ofgreater convenience, we have stopped the use of

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FIG. 1. Hfr strains of Salmonella typhimurium (shown outside the circle) and of S. abony (shown inside thecircle). The numbers in the circle represent time of entry for Hfr strains. The point of origin and orientation oftransfer of each strain is indicated by the arrow. For S. typhimurium the strain number (e.g., SR305) is given,and in Table 1 is found the Hfr designation (HfrA) and the genotype of each strain. In those cases in which F isinserted into a known transduction linkage group, the Hfr is displayed on an arc outside the main circle; thegenes shown on a cross-hatched arc are a P1-mediated transduction linkage group, but all other linkage groupsare for P22 phage. Where more than one Hfr strain number is shown in a gene interval (e.g., SA536, SA949,SA977, shown within a bracket), these represent independent isolates for which the point of origin is notproven different, though differences may exist. Not all strains with point of origin in the rfa genes (HfrKl andHfrK2, at 116 min) are shown. For more details see Tables 1 and 6 and reference 33. Discussion of experimentsrevealing point of origin and other properties of Hfr strains are in "Results" ("Hfr Strains of S. typhimurium"and "Hfr Strains of S. abony"). Only those genes whose positions are known with respect to the point of originof the Hfr strains are shown here; the complete linkage map is shown elsewhere (30).

616


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Millipore filters for the "contact period."SR305, the Hfr strain with which low frequencyof broth mating was observed earlier (31), inthis test as well gave increased mating onmembrane filters; this is a unique and unex-plained property of this strain. The matingmethod normally used in this report, describedin Materials and Methods, closely resemblesmethod 2; in those cases where Millipore filtersare used this is stated.

Stability of Hfr Strains During StorageThree Hfr strains of S. typhimurium, SR305,

SU418, and SA722, were stored by using thefour methods described in Materials and Meth-ods. At indicated intervals (Table 5), cultureswere grown from each of the three strains whichwere stored by each of the four methods, and50 single-colony isolates were assayed for fer-tility level by the replica plating method. InTable 5 is shown the percentage of high-fertil-ity isolates after storage of SA722. The per-centage of fertile lines of SA722 drops sharplyafter 36 or 70 days of storage in slant or stabculture, though a few fertile lines were re-covered even after 419 days of storage. Thelevel of fertility of the low-fertility isolates isconsistent with their being F+, though theyhave not been tested for transfer of F. Frozenor lyophil storage were the most satisfactory,with 90% of cells from lyophil storage retaining

TABLE 5. Percentage of high-fertility colonies ofSA722 (HfrKlO) after storage by different methodsa

Totaltime in Frozen Lyoph- Stab Slantstorage ilized(days)

1 100 100 100 1007 96 98 98 9814 84 NT° 80 8236 88 94 52 7270 72 88 36 6298 80 96 12 22132 78 92 6 10160 82 88 NT NT190 72 92 4 16231 82 90 4 8251 70 90 6 6419 62 92 4 6

aThe methods of storage used are described onpage 614 (Storage Methods). The percentages shownare based on tests of 50 colonies in each case. Thefertility of the colonies was determined by replicaplate methods, as described on page 614 (MatingMethods) and (Stability of Hfr Strains During Stor-age).

bNT, Not tested.

VOL. 36, 1972

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SANDERSON ET AL.

fertility, and the frozen stock dropping grad-ually to 62% in 419 days. The greater conven-ience of the frozen culture, coupled with theoccasional failure of aged lyophil stocks togrow, makes frozen stocks the method ofchoice, though Hfr strains should be kept inlyophil ampoules as well. Experience indicatesthat, with a few Hfr strains, all cells normallylose fertility after prolonged storage, even inlyophils.

Sources of the F+ StrainsThe F factor of E. coli K12 was transmitted

into S. typhimurium to produce SR297 F+,from which SR305 (HfrA) was isolated. It wasalso transmitted into another F- strain fromwhich SR315 (FS21) was isolated (43). SR297 isnot a good source of different Hfr strains, sincealmost all isolates were of HfrA type. ThereforeF was transmitted from SR297 into a consider-able number of F- auxotrophic strains of S.typhimurium by growth in mixed culture.Those F+ strains from which Hfr strains wereisolated are listed in Table 1.The F factor was similarly transmitted from

E. coli K12 into S. abony to produce theauxotroph SW1363 F+ and the prototrophSW803 F+ (20, 21); Hfr strains were isolatedfrom both of these. Hfr strains of type HfrHlwere present in high numbers among isolatesfrom SW1363 F+, and strains of type HfrH4were present among isolates from SW803 F+.The frequencies of recombinants from

crosses of five F+ strains of S. typhimurium toSU453, a recipient of S. typhimurium, aregiven in Table 6. SA622 and SA991 gaverecombinant frequencies typical of an F+strain, i.e., from 10-5 to 10- 6 recombinants perdonor cell for MetA+, Trp+, His+, and Xyl+,but SR297, SA27, and SA28 gave higher fre-quencies of recombinants, intermediate be-tween the usual frequencies for F+ strains andfor Hfr strains. S. abony F+ x S. abony F-crosses produced 10- 5 to 10- 6 recombinants perdonor cell, as did the nonhomologous crosses S.abony F+ x S. typhimurium F- and S.typhimurium F+ x S. abony F- (Table 7).

Hfr Strains of S. typhimuriumThe Hfr strains of S. typhimurium and of S.

abony listed in Table 1 were isolated from theseF+ strains as described in Materials and Meth-ods. The points of origin of the Hfr strains areillustrated in the partial linkage map of Fig. 1,with S. typhimurium strains outside and S.abony strains inside the circle. The S.

typhimurium Hfr strains are described below,in order of stock number, except that a group ofHfr strains with a similar point of origin issometimes discussed together. The S. abonyHfr strains are then treated in order of theirHfrH numbers, followed by a description of F'strains.SR305 (HfrA). Zinder (43) isolated SR305

from SR297 F+ and established that ilv is do-nated early; interrupted conjugation crosses(31) reveal this order: origin-ilv-metA-thr- --pyrE. An interrupted mating cross in whichthe cells were mated on a membrane filter (31)reveals (Fig. 2) that argF is transferred at about17 /2min, thi at 19 min.SR297 and SR305 carry an sfa, or sex-factor-

affinity locus, of the type described by Richter(27). SR305 is an unstable Hfr, reverting fre-quently to F+ state. However, both SR297 andany F+ strain resulting from reversion of SR305transfer their chromosome with the same pointof origin and orientation as SR305, though atlower frequency (Table 6). An interruptedmating experiment shows that ilv is a proximalgene for SR297 (Sanderson, unpublished data).In addition, over 100 Hfr strains were isolatedfrom SR297; all but one (SU436) appear identi-cal to SR305, and even SU436 reverts to an F+strain with the same orientation as SR297. IfSR305 is cured of F to yield the F- type, and isthen reinfected with F from an F+ strain, itregains the point of origin which is characteris-tic of SR297, and produces Hfr strains ofHfrA-type.SR315 (leu-256/FS21). SR315 transfers its

chromosome with the following orientation:origin-pyrE-trp-his-- -pyrC (28, 31, 43). Thestrain carries an F' factor (43), FS21, whichpromotes chromosome transfer with the abovepoint of origin. If SR315 is cured to the F- stateand then reinfected with wild type F, it has theF+ level of fertility, i.e., 10-5 to 10-6 recombi-nants per donor cell for all genes tested. How-ever, when FS21 was transferred into an F-strain, the resulting F' strain transferred itschromosome with the same fertility and pointof origin as SR315. FS21 must carry a portion ofthe chromosome of S. typhimurium from the42- to the 52-min region, thus promoting fre-quent integration of F at this point and hencechromosome mobilization, but the genes car-ried on FS21 are not identified.SU418 (HfrB2). First SU319, a line with

increased chromosome transfer, was isolatedfrom SR297. SU319 appears to transfer itschromosome clockwise from two points of or-igin, one between pyrE and ilv, as in SR297,

618 BACTERIOL. REV.


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F+, Hfr, AND F' STRAINS OF SALMONELLA 619

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620 SANDERSON ET AL. BACTERIOL. REV.

and one between his and trp (unpublishedM V

t ,s, data). Recombinants selected for genes in theo_4ooo = 4= ; t' 60- to 120-min interval seldom integrate donor

x X X Xx x genes from the 120- to 60-min region, and viceversa. In an effort to isolate the point-of-origin

cn_Nt|gX==in the trp-his interval, SU319 was crossed too) o> o o w .5 >, SU453 F- (trpB2 metA22 hisF1009 xyl-1x x x x 3 -e strA201) and Trp+ His+ recombinants were!q foCD con .= 0 selected; one of these recombinants was iso-

,;,, > X :E: lated as SU354 (HfrB2 metA22). SU354 wascrossed to SU208 (trpA8 proA26 F-), and a

-oo o o g X 3 = Trp+ Pro- fertile recombinant was designatedx x x x 4 .= SU418 (proA26 HfrB2); this strain also became

Oo s N > =; A = lysogenic for a phage of unknown source whichresembles and is designated P22. F. Casse

0000o o o .=,=(personal communication) found that, in a_ _ _ : E = g3cross with SU418 (HfrB2), 10% of recombinantsLo -x xq selected for the donor his+ allele integrate the

-=s3 <^, donor allele of chlC but that SA654 (HfrK9)does not transfer chlC as a proximal gene.

o000oo.o ° n Since chlC is jointly transduced with tdk,x.xxxX r < Xwhich is jointly transduced with trp, and since

cq -_0° tdk is not transferred distally by HfrB2, se-0;=2 0 quence of transfer for HfrB2 must be:

origin-chlC-tre-his- - -trp-tdk.z5EAg , Interrupted conjugation crosses with SU418.s=i;*; were reported earlier (31, 37). Selection for thes ° xO @ Q terminal gene, trp, in interrupted conjugationcoo produced the F prime factor FS71 trp and the

> > ~ -oa bDc. factors FS72, FS73, FS74, and FS75, each ofx x x x 0'WAC5 g' which carry trp-cysB-pyrF (32).

< < = s; SU436 (HfrB3). SU436 was selected from'W _ ° SR297 and used in early studies on the linkage

00o 0o o o : g <._map (Fig. 3) (31). SU436 frequently reverts tox x x x ,~ =a s:the F+ state, and since this F+ has the sfa locusv _ s _ E a= "Qr >characteristic of SR297, the F+ mobilizes the

chromosome clockwise from ilv. Thus, SU436c S must be single-colony-isolated immediately be-

fore use to maximize the proportion of Hfrcells, otherwise the genetic data it yields are

s+,-4= ,ambiguous; hence we now seldom use it for0. = g3 g bDgenetic analysis.-o W . SA458 (HfrK1-l), SA464 (HfrKl-2),

-0|<, SA537 (HfrKI-3), SA538 (HfrKl-4), SA539,,* <,, -8r(HfrKl-5), SA639 (HfrK1-6), SA642Q; CQ 6q = (HfrKl-7), SA967 (HfrKl-8). These strains0..° r transfer their chromosome with the following

eeOee=M S S rorientation: origin-pyrE-ilv-thr-- -cysE, with¢12 cJ cJ the F factor inserted in the rfa cluster of genes.

I= tZ.4HfrKl-1 to HfrKl-6 have been described (33),I 0E ;,> o so only a summary is given here.

0> -. The rough A (rfa) genes in S. typhimurium=i;> E v are required for the synthesis of the core region

oD t-r H o 0 - of the LPS, a component of the cell wall (23,-4 W.=2<:g ,"EH 36). Rough mutants make defective LPS and

cn co c9 UQ may be recognized by the chemotype of theirLPS (18, 19), by cultural characteristics, or,


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SANDERSON ET AL.

10SR305(HfrA)x SA1601 (F)

8 0 * ArgF+

o---3 Thi +

6

4

2

10

MATING TIME (I

FIG. 2. Interrupted conjugationtyphimurium strains SR305 (HfrAThe cells were mated on membranetransferred to broth, by techniques(31) and on page 615 (Method 5).interrupted by shaking the cells forMini-mixer.

most rapidly, by altered sensit(42). In HfrKl strains, the F facinto a cluster of the rfa genes N

transduced with cysE and pyriresults in chromosome transferof insertion of F, and simultane(phenotype, as determined by phThe hypothesis that F is insertgenes was confirmed by sever;tests, including the observatiHfrK1 strains transferred thoseest to pyrE on the proximalchromosome, while other genester were transferred late (33).An interrupted mating of SA9

(Fig. 4) indicates the followintransfer: origin-pyrE-ilv-metA-ingly, though pyrE is transferrecilv at 17 min, the total nunrecombinants after 60 min of r

(PyrE+/Ilv = 0.32), so that tUcrossed one another. Crossesstrains, which have different pwith SA572 F- did not show thefor example, a cross of SA536recipient showed normal kineticabout 5 min proximal to ilv, ac

the number of PyrE+ recombine

than the number of Ilv+ recombinants. Thesedata indicate that the low frequency of integra-

o tion of PyrE+ in SA464 is due to the point oforigin being very close to pyrE, with resulting

/ low frequency of integration of PyrE+, which/ is on the proximal end of the chromosome. This

j/ explanation was invoked when similar observa-13/ tions of low incorporation of immediately prox-

/ / imal markers were made in E. coli (3a, 15, 26).In most instances, the insertion of F into the

l° chromosome to produce an Hfr strain does notalter the phenotype, other than for properties

// associated with fertility. However, in E. coli, F// can insert into the lac operon (R. Curtiss,

Bacteriol. Proc., p. 30, 1964), and insertion of F/0/ocan produce resistance to phages Ti or T6 or Ti// and T5 (2). Thus it appears that F can insert at9 any point in the continuity of the chromosome.LL Occasionally this insertion may be within a20 30 gene or operon, preventing transcription orM NUTES) translation of genes; this may result in observa-

ble changes in phenotype, as in the casescross ot the S. reported above.x SA1601 F-. The pattern of phage sensitivity of these

e filters and then HfrKl strains was tested at the University ofdescribed earlier Calgary and confirmed by B. A. D. Stocker atThe mating was Stanford University. The different HfrKl1 mm in a Fisher strains had phage sensitivity approximating

the pattern R-res-2, or R-res-1 (characteristic.livit to phages of rfaG mutants), or epi-1 (characteristic ofirvity to phages rfaG mutants), or R-sens, the phage patternstor has inserted .'.hichis jointly having been established by Wilkinson et al.whflc), Al2 rPLny (42). Each of these patterns of phage resistance

from the pointously in the Rfalage sensitivity.ted into the rfaal independention that some

rfa genes clos-part of their

in the rfa clus-

164 x SA572 F-ig chromosomemaiB. Surpris-I at 12 min, andnber of PyrE+mating was lowhe entry curvesof other Hfr

)oints of origin,same anomaly;with the same

-s; pyrE entereds in SA464, butants was greater

3'

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z

m O 1.8

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SU436(HfrB3)x SU280(F-

----* ArgE+ /

o---o SerA+/

//A ~Tyr+ 0

. 1

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v /1

,5

10 20 3 4 50

10 20 30 40 50

i_

MATING TIME (MINUTES)FIG. 3. Interrupted conjugation cross of SU436

(HfrB3) x SU280 F-, both strains of S. typhimur-ium. The cells were mated on membrane filters as

described in the legend to Fig. 2.

LLna-UAI-

<C xZ- COU

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622 BACTERIOL. REV.

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z ~~XBAD,/t- I /L 2u) 20 _ .Iliv+

Z n o---OPyrEE

z MetA[0s15 ; A---AmatlB+

Iuo 10 /

0 ~ ~ 2 30 40 5

0

tyhmruC trsS44(fr12 A7 F- .

w

m-

M: 5/z,

10 20 30 40 50

MATING TIME (MINUTES)

FIG. 4. Interrupted conjugation cross of the S.

typhimurium strains SA464 (HfrKl-2) x SA572 F

The cells were mated in broth by using techniquesdescribed on page 612 (Mating Methods) and in the

footnote to Table 6. The mating was interrupted at

the times indicated on the graph by blending the

cells for 20 sec in the Micro-jar of a Waring blend or.

The cells were then plated on selective minimalmedium.

is characteristic of mutants in the rfa cluster of

genes. The phage-sensitivity phenotypes are

shown in Table 1, and the phage patterns ofrepresentative strains of each class are in Table3.

SA486 (HfrK3). In a cross between SA486

and SA571,10;2 to 10-3 recombinants per

donor cell were obtained for genes in the malAto metA region, 10-4 to 10-6 for his and trp

(Table 6). Plate mating experiments indicate

that the point of origin is in the 95-mmn region

of the chromosome, so SA486 was mated in

broth for 60 min with a series of singly auxo-

trophic F strains, and plated on minimal

medium selective for prototrophic recombi-

nants; the numbers of recombinants observedper 0.1 ml of mating mixture were as follows:

metC193, 20,000; argE116, 30,000; aroC74,

9,000; aroB36, 20,000; cysG382, 10,000; argG 10,

4,000; pyrE125, 3,000; cysCD519, 0; lysA8, 0;

serA 13, 0; argB69, 0. An interrupted conj'uga-tion cross of SA486 x argEll16 F (Fig. 5) shows

that argE+ is transferred with linear kinetics

after 12 mmn of mating. Determination of the

point of origin is complicated by the fact that

SA486 has the serAl3 mutation; the low fre-

quency of SerA+ recombinants observed in

crosses to serA 13 does not demonstrate that

serA 13 is transferred late. Crosses using a serA +

revertant of SA486 have shown that serA istransferred early (K. Chater, personalcommunication).SA534 (HfrK4). A cross to SU453 F- pro-

duced 3 x 10-4 Trp+ recombinants but only10-5 MetA+ recombinants per donor cell (Ta-ble 6). A series of plate mating experimentswith singly auxotrophic F- strains gave highnumbers of recombinants with pyrB and pro

2.5r

LLa-

z

Z

m

o xC

LL

crLiJmD

z

2.0O

1.5

1.0

0.5 F

SA486 (Hf rK3)x argE116 (F-)

ArgE'

i/ I I I10 20 30 40


50

FIG. 5. Interrupted conjugation cross of the S.typhimurium strains SA486 (HfrK3) x argE116 F-;methods were as described in the legend of Fig. 4.

40r

olr

z<:m

- om v

2 x0 _

crLL)

-0C

a:

z

SA535 (HfrK5 )x SU674 (F- )

*5-. Met G+

PPurG+

I

1 0 20 30 40 50

MATI NG TIME (M NUTES)FIG. 6. Interrupted conjugation cross of the S.

typhimurium strains SA535 (HfrK5) x SU674 F-;methods were as described in the legend of Fig. 4.

VOL. 36, 1972 623

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624 SANDERS

mutants and low numbers with ilv, metA, andpurA mutants. These data indicate the follow-ing chromosome transfer: origin-pyrB-thr-trp-- -purA.SA535 (HfrK5). An interrupted conjuga-

tion cross with SU674 F- (Fig. 6) revealed thatmetG+ is transferred by SA535 at 6 min andpurG+ at 20 min, while the number of His+recombinants is still low at 50 min. ThereforeSA535 transfers its chromosome as follows:origin-metG-aroD-purG---his. From SA535an F' his factor, FS400, has been isolated (39).SA536 (HfrK6), SA949 (HfrK7), and

SA977 (HfrK8). These three Hfr strains alltransfer clockwise, O-xyl-cysE-ilv-- -malA.This point of origin was determined in crossesto SA571 and SA570; all three independentlyderived strains produce 10- to 10- 2 recombi-nants per donor cell for proximal genes such asxyl, but only 10-5 to 10-6 recombinants fordistal genes such as malA (Table 6). An inter-rupted conjugation cross of SA536 x SA570 F-(Fig. 7) gave linear entry curves (14, 33).

In the hope of isolating Hfr or F' strains, allthree Hfr strains were crossed to SA571 F- in amating interrupted at 60 min, and recombi-nants for the distal gene on the Hfr, malA+,from each of the crosses were patched toselective medium and replica plated to test forfertility and genotype. Among 20 to 25 MalA+recombinants from each cross, 90 to 100% alsowere donor type for proximal genes(Xyl+PyrE+Ilv+MetA+), and all were recipienttype for genes in the middle portion(His -Trp- ). In addition, none gave detectable

20

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10 20 30 40 50

MATING TtME (MINUTES)FIG. 7. Interrupted conjugation cross of the S.

typhimurium strains SA536 (HfrK6) x SA570 F-;methods were as described in the legend of Fig. 4.

'ON ET AL. BACTERIOL. REV.

chromosome transfer, except one recombinantfrom SA536 which had the same pattern ofchromosome transfer as its donor parent. Thefailure to detect His+ and Trp+ recombinantsselected for a distal gene was surprising, and itis noted in some other Hfr strains.SA540 (HfrK2-1), SA640 (HfrK2-2), and

SA653 (HfrK2-3). These strains transfer theirchromosome as follows: origin-cysE-xyl-malA---pyrE, with F inserted into the rfacluster of genes, but in the opposite orientationto that observed in HfrK1 strains (Table 6;Fig. 8; reference 33). The HfrK2 strains are allRfa in phenotype, as in the HfrKl strains:HfrK2-1 and HfrK2-2 have the phage-sensi-tivity patterns R-res-1 and R-res-2, respec-tively (33).SA654 (HfrK9). This strain, which resem-

bles HfrB2, transfers his as a proximal markerand trp as a distal gene, but it differs in that ittransfers tre late.SA722 (HfrK1O). A cross to SA571 F- gave

high frequency of recombination for ilv but lowfrequency for metA (Table 6). An interruptedconjugation cross with SU633 F- revealed thatilv + is transferred after 6 min of mating, with 2x 10-2 Ilv+ recombinants per donor cell by 60min, whereas the number of MetE+ recombi-nants at 60 min is only 10 per donor cell (Fig.9). Of 85 Ilv+ recombinants, all retained therecipient-type metE- allele. Transfer of thegene cya, which is known to be jointly trans-duced with both ilv and metE, in the orderilv-cya-metE, was tested in a cross of SA722 toDB99 F -; Ilv + recombinants occurred at afrequency of about 10-3 per donor cell, whileMet+, Pur+, and Pro+ recombinants were 10-5to 10-6 per donor cell. Of 25 Ilv+ recombinantsall were Met-Pur-Pro-Cya-. These data indi-cate that cya is transferred as a distal gene bySA722, which must have the following transfersequence: origin-ilv-pyrE-xyl-- -metE-cya.SA828 (HfrK25). Following selection from

the F+ for high frequency of chromosometransfer, the strain was selected for resistanceto phage FO; thus, the strain is a rough mutant.It transfers with the orientation trp-his-purC- - -gal (Table 6).

SA949 (HfrK7). Origin and direction oftransfer are very similar, if not identical, toSA536 (see above).SA952 (HfrK14), SA967 (HfrKl-8), and

SA975 (EfrK13). Crosses to SA571 F- in-dicated that this strain transfers its chromo-some with the following orientation:origin-pyrE-ilv-metA-trp-his-malA-xyl (Table6). This resembles strains of the HfrKl typewhich arise owing to insertion of F into the rfa

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- x y l +

Ej---o MaIA+A H-ilisF+,TrpB

I -o

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ad

10 20 30 40 50MATING TIME (MINUTES)

FIG. 8. Interrupted conjugation cross of the S.typhimurium strains SA540 (HfrK2-1) x SA571 F-;methods were as described in the legend of Fig. 4.

genes and which result in the Rfa phenotype(see SA458, above). However, SA952 has thephage sensitivity of a smooth strain, thusresembling SA991, the F+ strain from which itoriginates. Both SA967 (HfrK1-8) and SA975(HfrK13) give the same transfer pattern incrosses to SA571 (Table 6), and both originatefrom the same F+ strain; SA967 has the"rough-sensitive" pattern of phage sensitivity,whereas SA975 is smooth (Table 3). All threestrains were crossed to SA570 F-, with selec-tion for Xyl+, CysE+, Ilv+, and MetA+ recom-binants (Table 10). These data confirm that allthree strains transfer ilv at high frequency andxyl at low frequency, and suggest that cysE istransferred early by SA975, but late by SA952and SA967. From these crosses, MetA+ re-combinants and CysE+ recombinants wereselected, streaked for single-colony isolation,and tested for auxotrophic phenotype, strep-tomycin sensitivity, phage sensitivity as anindicator of the rfa allele, and for fertility(Table 11). In the cross with SA952, among 12MetA+ recombinants, 11 also integrated thedonor ilv+ allele; no other donor alleles weredetected. Among recombinants selected forCysE+, a terminal marker, 6 of 12 recombi-nants integrated the donor allele, 9 of 12integrated the donor rfa allele, 8 of 12 the donorilv allele, and 9 of 12 the donor fertility level;very few other donor alleles were integrated.These data do not permit unequivocal locationof the point of insertion of F, but since allMetA+ recombinants are recipient type for rfa,F is assumed to be inserted between rfa and ilv,

transferring ilv first. Other data (Table 6)showed that pyrE is transferred proximally, soSA952 transfers as follows: origin-pyrE-ilv-metA---rfa.

In initial tests, SA967 was resistant to phageFO, sensitive to Ffm, so SA967 was distinguish-able in its pattern of phage sensitivity from therecipient, SA570. All 12 MetA+ recombinantswere donor type for ilv but recipient type forRfa. This strain is classified as an HfrKl-typeand given the isolation number HfrK1-8. In alater test, SA967 is sensitive to phage FO,exhibiting the "rough sensitive" phage pheno-type (Table 3).From the cross SA975 x SA570, all 12 MetA+

recombinants are donor type for cysE, rfa, andilv; this confirms that the chromosome of thisstrain transfers as follows: origin-cysE-pyrE-ilv---xyl. Two genes concerned with manni-tol fermentation, mtlA and mtlB, are locatedbetween cysE and xyl, according to P22-medi-ated transduction crosses (34). In crosses toSA787 F-, a strain carrying a mtlA mutation,the frequency of Mtl+ recombinants was about10- per donor cell for all three Hfr strains,suggesting that mtl is transferred as a distalgene. However, based on fermentation testson Difco Deoxycholate Agar plus 2% mannitol,SA975 is a mannitol nonfermenting strain,though SA991, the F+ from which SA957 origi-nates, and SA952 and SA967, Hfr strains iso-lated at the same time as SA975, are all man-nitol-fermenters. The coincidence of insertionof F in the cysE-xyl interval, which includes

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FIG. 9. Interrupted conjugation cross of the S.typhimurium strains SA722 (HfrKlO) x SU633 F-;methods were as described in the legend of Fig. 4.

A

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TABLE 8. Fertility of Hfr strains of Salmonella abony and S. typhimurium with points of origin betweenmin 50 and min 90 in crosses with S. abony and S. typhimurium recipientsa

No. of recombinants per donor cell"

Donor species strain ... S. abony SW1403 S. abony SH465 S. abonv SH462 S. typhimurium SU418HfrH2 HfrH5 HfrH12 HfrB2

Recipient species .... TM abony TM abony TM abony TM abony

Mutant locus in re-cipient strain

metA 128d <10-6 <10-6 1 X10-l 1 X 10-6 1 x 10-4 2 x 10- 6arg 1 X 10-5 3 x 10-6 3 x 106 1 X 10-6ilv 122 <10-6 <10-6 <10-6 <10-6 4 x 10-6 1 X 10-4 1 X 10-4pyrE125 116 1 x 10-6 5 x 10-5argE8 102 2 x 10-5 <10-6 <10-6 4 x 103serA 95 5x 10-5 2x 10' 4x 10-4aro 4 x 10-6 3 x 10-' 1 X 10-4purC7 78 5x 10-4 3x 10-5 2x 10-4 4x 10-3his 65 3 x 103 6 x 10-3 1 X 10-3 2 x 10-2 2 x 10-3 1 X 102 7 x 10-3trp 52 2 x 10-4 5 x 10-5 2 x 10-5 1 x 10-5gal 33 1 x 10-4 1 X 10-6' 2 x 10-6purE 19 <10-6 <10-6 2 x 10-6pro 10 <10-6 1 X 10-4 <10-6 <10-6 <10-6 5 x 10-6 1 x 10-6leu 3 2 x 10-4 <10-6 3 x 104 6 x 10-5pyrB 0 8 x 10-6 1 X 10-4

a Mating methods are described in Table 6, footnote a, and on page 612 (Mating Methods).b In all crosses the recipient is auxotrophic for a mutation in the gene shown in the left-hand column, and

recombinants are selected in a cross to a donor having the wild-type allele of this gene.CTM, typhimurium.d The number shown with the mutant locus of the recipient indicates the location in minutes of the mutant

gene on the S. typhimurium linkage map.

the mtl genes, with change to a mannitol-non-fermenting phenotype suggests, but does notprove, that F may be inserted into the mtlgenes.SA955 (HfrK20) and SA965 (HfrK17).

Both of these strains produce over 10- 2 Pro+recombinants per donor cell and appear totransfer counterclockwise, with trp as a distalgene (Table 6). These Hfr strains, and alsoSA536, were crossed to SA1475 F-, to SA1476F-, and to SL680 F-; MetA+, Trp+, Gal+,PurE+, Pro+, Ilv+, and PurC+ recombinantswere selected (Table 12). The data indicate thefollowing points of origin: SA955(HfrK20)-origin-purE-pro-leu-- -gal; SA965(HfrK17 )-origin-pro-metA-ilv- - -purE. Thepoint of origin of SA536 is already known fromother data and confirms the location of thegenes. An interrupted conjugation cross ofSA955 x SA1476, with selection for PurE+Str-r and Pro+ Str-r recombinants (Fig. 10),revealed that purE enters the recipient at ca.16 min, pro at ca. 24 min; the 8-min interval issimilar to that previously reported (31).SA962 (HfrK16). This strain transfers his

and tre as proximal genes and trp as a distalgene (Table 6).

SA965 (HfrK17). See SA955, above.SA966 (HfrKI9). Crosses to SA571 and

SL680 suggest the following chromosome trans-fer: origin-metA-ilv---pro. Since SA966 isisolated from SA991, which is of genotypethrA49 leuCD39 ara-7, selection for Thr+,Leu+, and Ara+ recombinants was not at-tempted. To determine if thr, leu, and ara aretransferred early, SA966 was crossed to SA1475F-, and recombinants were selected on MMcontaining threonine plus leucine, with strep-tomycin in the medium to prevent growth ofthe donor (Table 6). Ilv+, Met+, and Gal+recombinants were tested for their nutritionalphenotype and for fertility (Table 13). Only afew of the MetA+ recombinants carry any ofthe distal genes; none carry the donor alleles forThr-, Leu-, or Ara-. Twelve of 24 Ilv+ recom-binants are Met+, but few integrate distalgenes from the donor. Among 23 recombinantsfor Gal+, a gene near the distal end of thechromosome of SA966, about one-half (9 to 14)carry the donor alleles for genes close to theproximal end, from metA to malA, and twohave the donor alleles for Thr-, Leu-, and Ara-phenotype; these data indicate that thr, leu,and ara are transferred as distal genes. Subse-

626 BACTrERIOL. REV.


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SANDERSON ET AL.

quently, SA966 and some other Hfr strainsderived from SA991 were found to have losttheir requirement for threonine (Table 1, foot-note d). Since Thr- recombinants were re-covered from the above cross, the Thr+phenotype of SA966 is probably due to asuppressor mutation. The data indicate thatSA966 transfers as follows: origin-metA-ilv-pyrE---gal-(leu-ara-thr). Based on platemating methods, recombination with purA58

TABLE 10. Number of recombinants per donor cell incrosses of Salmonella typhimurium Hfr strains to S.

typhimurium SA570 F- a

Donor Selected genestrain Xyl+ CysE* Ilv + MetA+

SA952 <10-6 2x10-5 8x 10- 2 7x 10- 2

SA967 <10-6 <10-6 6 x 10- 2 2x 10- 2

SA975 <10-6 9 x 10-2 9 X 10- 2 6 x 10- 2

aThe cells were mated in broth for 60 min bytechniques described in Materials and Methodsand in footnotes to Table 6.

was very high, and with pyrB96 it was very low.An interrupted conjugation cross of SA966 x

SA1515 (purA58 ara-9) yielded 10- 1 PurA+recombinants per donor cell after 60 min ofmating but only 10-' Ara+ recombinants (Fig.11). It is not possible to determine the time ofentry of purA, for the kinetics of transfer ap-

pear exponential rather than linear. In the in-terrupted conjugation cross of SA966 withSA1602 F- (Fig. 12) and with SA1475 F- (un-published data), exponential transfer kineticswere again observed. The order of gene trans-fer agrees with the order based on 60-min mat-ings, plated without interruption, i.e., origin-metA-ilv-cysE-xyl (Table 6), but the apparenttime of entry of ilv, cysE, and xyl is late andrecombinants are infrequent. This strain isapparently not useful for time-of-entry studies,at least under the conditions used in our

crosses.

SA967 (HfrK1-8). See SA458 and SA952,above.SA969 (HfrKl5). A cross to SA571 F-

indicated that ilv is transferred as a proximalgene and metA as distal gene (Table 6). Platemating experiments using recipients with mu-

TABLE 11. Genotype of recombinants from crosses of Salmonella typhimurium x S. typhimurium SA570 F:recombinants are selected from the crosses recorded in Table 10

Donor Selected xyl cysE rfaa ilv metA thr| leub trp his strb| Fertil- Fre-

SA952 MetA+ Od 0 0 1 le 0 0 0 0 0 0 11y0 0 0 0 1 0 0 0 0 0 0 1

CysE + 1 le 1 1 0 0 0 C 0 0 1 4o le 1 1 U 0 0 c 0 0 1 20 le 1 1 1 0 0 0 0 0 1 10 le 1 0 0 0 0 1 1 0 1 10 le 0 0 0 0 0 0 0 0 0 11 le 0 0 0 0 0 0 0 0 0 11 le 0 1 0 0 0 0 0 0 0 10 ie 1 0 0 0 0 C 0 0 1 1

6 12e 9 8 1 0 0 1 1 0 9 12

SA967 MetA+ 0 0 0 1 le 0 0 C 0 0 0 12

SA975 MetA+ 0 1 1 1 le 0 0 0 0 0 0 12

a SA952 and SA975 are smooth strains and hence are sensitive to phages P22 and FO, resistant to Ffm.SA967 has phage sensitivity characteristic of rfa mutants, being sensitive to phage Ffm but resistant to phagesP22 and FO. SA570 has the phage-sensitivity pattern termed "rough-sensitive" associated with the rfa allele.All three phage sensitivity pattems could be recognized among the recombinants.

b The recipient alleles of thr+, leu+, and strr were selected.c The fertility level of the recombinants was established by replica-plating the recombinants onto a plate of

minimal medium on which cells of an auxotrophic recipient strain had been spread, and then incubating andobserving the frequency of recombination.

d 0, Recipient type; 1, donor type.e Selected donor allele.

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TABLE 12. Number of recombinants per 0.1 ml ofmating mixture in crosses of Salmonella

typhimurium Hfr strains to S. typhimurium F-strains, after 60 min of matinga

Donor strain

Recipientstrain Recom- SA955 SA965 SA536

selected (HfrK20) (HfrK17) (HfrK6)

SA1475 MetA+ 12,200 9,000 100,400Gal+ 45 40 42,500TrpE+ 91 63 3,500

SA1476 PurE+ 21,400 3 3,000Pro+ 8,100 6,500 2,200

SL680 Pro+ 10,900 17,900 26,000Ilv+ 16 394 106,800PurC+ 2 37 800

aThe matings were done in broth by using tech-niques described on page 612 (Mating Methods) andin footnotes to Table 6.

tations in the ilv-metA interval (122 to 128 minon the linkage map) suggested that thi, argF,metF, and ilv are transferred as proximalgenes, whereas metA and purD are transferreddistally. This was confirmed as follows. Fromthe cross SA969 x SA1601 (argFl18 thi-36 F-),the frequency of ArgF+ and Thi+ recombinantswas 10- 2 and 3 x 10- 3, respectively; since mostrecombinants were donor type for both genes,both are transferred as proximal genes. In thecross SA969 x SA1504 F-, MetE+ recombi-nants were detected early, whereas PurD+recombinants were very infrequent, even after60 min of mating (Fig. 13), indicating thatmetE is a proximal gene and purD a distalgene. Based on P22-mediated transduction, thegene order is metA-purD-thi-argF-metB (30);the insertion of F must be between purD andthi, so the order of transfer is:origin-thi-argF-ilv--- -purD.SA970 (HfrK11) and SA978 (HfrK12). In

crosses of these two Hfr strains to SA571 F-,there are 10-2 to 2 x 10-l recombinants perdonor cell for proximal genes such as pyrE, xyl,and malA, but 10-4 to 10-5 recombinants perdonor cell for a distal gene, ilv (Table 6). Thusthese strains, derived from different F+ strains.both transfer counter-clockwise from a point-of-origin at 117 to 122 minutes,O-pyrE-cysE-xyl-- -ilv. An interrupted conju-gation cross, SA978 x SA571 F-, suggests thatpyrE+ is transferred at 10 min, and xyl about 1min later (Fig. 14).SA970 and SA978 were each mated with

SA571 F- for 60 min and plated on selectivemedia without interruption; recombinantswere then selected and tested for genotype andfor fertility (Table 14). If a gene close to theorigin, such as xyl or malA, is selected, there isa high frequency of integration of proximalgenes such as pyrE (from 25 to 60%), interme-diate frequency of integration of trp and his,whereas characters transferred distally, such asmetA, ilv, and fertility, are not detected. Whena late gene such as metA or ilv is selected, ahigh proportion of the recombinants are fertile,indicating transfer and integration of F, andmany also integrate the proximal genes pyrE,xyl, malA. In tests for fertility, all the fertilerecombinants give many pyrE and cysE recom-binants but few ilv or metA recombinants,suggesting that they are Hfr rather than F'types. Surprisingly, the genes which are trans-ferred in the middle of the chromosome, trpand his, are not integrated in recombinants fordistal genes such as metA or ilv. Among 25Trp+ recombinants, none carried any proximalor distal donor alleles except for his+.SA975 (HfrK13). See SA952.SA977 (HfrK8). See SA536.SA978 (HfrK12). See SA970.

Hfr Strains of S. abonyThe genotype, source, and properties of ge-

netic transfer of the Hfr strains of S. abony aredescribed in Table 1, and the point of origin is

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FIG. 10. Interrupted conjugation cross of the S.typhimurium strains SA955 (HfrK20) x SA1476 F-;methods were as described in the legend of Fig. 4.

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TABLE 13. Phenotype of recombinants from a cross of Salmonella typhimurium SA966 (HfrK19) x S.tyr himurium SA1475 F- a

Fertil-MetA Ilv PyrE Xyl MalA Str HisF TrpE Gal Leu Ara Thr ity Frequency

lb 1 1 1 0 0 0 0 0 NT 0 NT 0 1lb 1 0 0 0 0 0 0 0 NT 0 NT 0 2Ob0 0 0 0 0 0 0 0 NT 0 NT 0 22/25

1 lb 1 1 0 0 0 0 0 0 0 0 0 11 lb 1 0 0 0 0 0 0 0 0 0 0 1O lb 1 1 0 0 0 0 0 0 0 0 0 21 lb 0 0 0 0 0 0 0 0 0 0 0 100 lb 1 0 0 0 0 0 0 0 0 0 0 20 lb 0 0 0 0 0 0 0 0 0 0 0 8/24

1 1 1 1 1 0 0 0 lb 0 0 0 0 50 1 1 1 1 0 0 0 lb 0 0 0 0 11 1 0 1 1 0 0 0 lb 0 0 0 0 11 0 1 1 1 0 0 0 lb 0 0 0 0 11 1 1 1 0 0 0 0 lb 0 0 0 0 10 0 1 0 0 0 0 0 lb 0 0 0 0 11 0 1 1 1 0 0 0 lb 0 0 0 0 11 0 1 0 0 0 0 0 lb 0 0 0 0 11 0 0 0 1 0 0 0 lb 0 0 0 0 11 0 0 0 0 0 0 0 lb 0 0 0 0 11 1 1 1 1 0 0 0 lb 1 1 1 0 11 0 0 0 0 0 0 0 lb 1 1 1 0 10 0 0 0 0 0 0 0 lb 0 0 0 0 7/23

'Mating methods are described in Table 6, footnote a, and on page 612 (Mating Methods). Recombinantswere transferred to a master plate of supplemented minimal medium and tested for nutritional phenotype byreplica plating onto supplemented minimal medium; for fertility, recombinants were tested by replica platingonto a lawn of F- cells as described in Materials and Methods.

bIndicates the selected donor allele. 1, Donor type phenotype; 0, recipient type phenotype; NT, not tested.

SA966 (HfrKl9 )x SA1515(F-)

APurA0- - - A ra +

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o---C Ilv+h- - CysE+L---A X y L +

1 0 20 30 40 50 60

MATING TIME (MINUTES)FIG. 11. Interrupted conjugation cross of the S. FIG. 12. Interrupted conjugation cross of the S.

typhimurium strains SA966 (HfrK19) x SA1515 F-; typhimurium strains SA966 (HfrK19) x SA1602 F;methods were as described in the legend of Fig. 4. methods were as described in the legend of Fig. 4.

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illustrated in Fig. 1. Frequencies of recombi-nants with these strains as donors are given inTables 7, 8, and 9. Both S. abony and S.typhimurium recipients were used; recombi-nation is more frequent in the homologous com-bination with S. abony recipients. Some S.typhimurium donors are included for compari-son; they also give more recombinants in thehomologous combination.HfrHI. This is the only Hfr of S. abony

in which F-factor integration is so stable that itis not possible to demonstrate a transmissible Ffactor, in which all recombinants for early genesare still F-, and in which the Hfr character is

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stable on storage (20). HfrHl transfers pyrBearly and purA late (31), and hence the order oftransfer is origin-pyrB-pro-- -purA (Table 7).It is lysogenic for PKy1O3, the attachment siteof which is close to purE; since the prophage istransferred early, zygotic induction interfereswith genetic analysis in the first quarter of thelinkage map, for which this Hfr is otherwisevaluable. Cured derivatives were prepared (1),and useful markers were introduced into thissegment of the HfrH1, such as purE66 from S.typhimurium, oafR+ (determining a stable an-tigen 122) from S. enteritidis (22), and rftA(determining the synthesis of the Ti anti-

15rSA969 (Hf rK15)x SA1504 (F-)

8 M et E+a---t3 PurD +

10 20 30 40 50 60


FIG. 13. Interrupted conjugation cross of the S.typhimurium strains SA969 (HfrK15) x SA 1504 F-;methods were as described in the legend of Fig. 4.

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FIG. 14. Interrupted conjugation cross of the S.typhimurium strains SA978 (HfrK12) x SA571 F-;methods were as described in the legend of Fig. 4.

TABLE 14. Number of recombinants with alleles from the donorparent among recombinants from crosses ofSalmonella typhimurium Hfr strains with S. typhimurium SA571 F- a

HfrSelected donorl No tested pyrE GGene testedHfr Seleedor No.tested1gene ~~~~pyrE xyl malA his trp jmetA ilv Fertility

SA978 Xyl+ 50 30 50b 113 111 4 0 0 0SA978 MalA+ 47 13 26 47b 6 1 0 0 0SA978 MetA+ 50 11 10 5 0 0 50b 15 4

SA970 Trp+ 25 0 0 0 1 25b 0 0 0SA970 Met+ 21 4 2 2 0 0 21b 5 2SA970 Ilv+ 9 4 1 1 0 0 3 9b 9

a Mating methods are described in Table 6, footnote a, and on page 612 (Mating Methods). Recombinantswere transferred to a master plate of supplemented minimal medium and tested for nutritional phenotype byreplica plating onto supplemented minimal medium; they were tested for fertility by replica plating onto alawn of F- cells as described in Materials and Methods.

b Selected donor allele.

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gen-35) from S. paratyphi B, Ti. In thesemanipulations, the HfrHl strain was used asan aeration F- phenocopy. Later, several moreHfr strains were isolated from SW1363 F+ andappeared to have the same origin and directionof transfer as HfrHl, suggesting the presence ofa sfa locus in SW1363 F+The original HfrHl strain SW1444

(=-SW1391 in Makelai [20]) was auxotrophicand streptomycin resistant. A prototrophicstreptomycin-sensitive derivative was isolatedafter mixed culture with a prototrophic F- S.abony strain to give SH81 (24). It was firstcalled HfrH6 but appears identical withHfrHl. This strain was more suitable thanSW1444 for interrupted mating crosses, be-cause streptomycin, which could be used forcounter selection against the male parent,prevented plate mating. An interrupted conju-gation cross with SH81 (HfrH1; Fig. 15) showsthe time of entry of pro to be about 18 min andof gal to be about 30 min. SH81 has an rfb mu-tation; the phage pattern (Table 1) is slightlydifferent from the "FO-sensitive" pattern ofrfb mutants of S. typhimurium and probablyreflects intrinsic differences between thesespecies. An F' derivative, FS59, has been iso-lated from HfrHl (24).HfrH2 (SW1403). This strain was isolated

from the same auxotrophic, streptomycin-resistant strain as HfrHl (20). Its F is trans-missible, and most recombinants are F+. Uponreinfection with F after loss of F on storage, itregains its original fertility. It has acquired a

second A-type prophage as well as PKylO13, andits phage sensitivity is typical of an A-lysogenicsmooth strain. It transfers its chromosome asfollows: origin-purC-his-- -argE (Table 8); theprophages do not interfere with genetic analy-sis of proximal genes. The aro mutation ofSW1403 is transferred early. Recently, an F'has been isolated from HfrH2 by Wyche et al.(Abstr. Annu. Meeting Amer. Soc. Microbiol.,1972).HfrH3, SW1452.- This strain transfers its

chromosome in the order origin-gal-pro-- -trp(20). It has low fertility, giving only 2 x 10-4recombinants per donor cell, compared with10- ' to 10- 2 for the more fertile Hfr strains. Thedonor properties of this strain are too unstablefor use in genetic analysis, but a more stablereisolate, SH566, has been used in interruptedconjugation (Fig. 16). Another reisolate fromSW1452 was found to be of the type of HfrH4(31).HfrH4 (SW1446). This strain transfers its

chromosome as follows: origin-metA-ilv---pyrB (Table 7). An interrupted mating crossis shown in Fig. 17. In HfrH10, also isolatedfrom SW803 F+, the point of origin resemblesand may be identical to that of SW1446.Several other Hfr isolates from SW803 F+appeared to be of the same type, suggestingthe presence of a sfa locus in the F+ strain(20).

HfrH5 (SW1462) (20). SW1462 transfers itschromosome as follows: origin-trp-his-- -purB(Table 8); Fig. 18 shows an interrupted mating

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abony strains SH566 (HfrH3) x SH632 F-; methodswere as described in the legend of Fig. 4.

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FIG. 17. Interrupted conjugation cross of the S.abony strains SW1446 (HfrH4) x SH632 F-; meth-ods were as described in the legend of Fig. 4.

cross. This strain has low fertility and fre-quently loses fertility, so it is not as useful forgenetic analysis of genes in the his region as isHfrH2.HfrH7 (SH475). This strain transfers its

chromosome as follows: origin-ilv-pyrB---pyrE (Table 9). Since its donor properties arerelatively stable, it is useful for analysis of the120- to 130-min segment of the chromosome.HfrHlO. See HfrH4.HfrH12 (SH671). This strain injects in the

order origin-purC-his-- -serA (Table 8). Thechromosome transfer resembles that of HfrH2,but the points of origin of HfrH2 and HfrH12appear different; HfrH2 is the more stable anduseful strain. This strain and its derivateSH462 have the "anomalous FO-sensitive"phage sensitivity pattern (see Table 3), whichis probably due to a rfx mutation.

F' Factors Carrying Salmonella GeneticMaterial

Though the number of Hfr strains availablein Salmonella is considerable, and many ofthese have been isolated for some years, F'factors carrying Salmonella genetic materialare available for only a few regions of thechromosome. The F' factors are listed in Table1 and discussed below. They are generallydesignated as FS, with the S referring toSalmonella genetic material (although F or FThas been used instead of FS in earlier reports).

S. typhimurium Genetic MaterialFS71 to FS75 factors carry trp in the case of

FS71 and trp cysB pyrF in the case of FS72 toFS75. All were isolated from a cross of SU418(HfrB2) with selection for a terminal marker,trp+, following interruption of mating after 60min (32).FS76 and FS77 are F' factors isolated after

plate-mating crosses of SA464 (HfrKl-2) xSA508 F-, with selection for a terminal marker,cysE+. Of ca. 100 such recombinants, abouthalf were HfrKl type, about half were F-, buttwo gave high-frequency transfer clockwisefrom cysE, segregated CysE- recombinants,and transferred the capacity for chromosometransfer and segregation to all CysE+ recombi-nants (Sanderson and Saeed, unpublisheddata). FS76 carries xyl-cysE-rfa-pyrE, whileFS77 carries only cysE.The FS400 his factor was isolated by Voll

(39) from a cross of SA535 (HfrK5) by earlyselection for a terminal marker, his+, followinginterrupted mating. The factor was transferredinto an E. coli strain for maintenance. FS400carries rfb gnd his.

S. abony Genetic MaterialThe FS59 factor was isolated from S. abony

HfrHI when looking for "transmissible Hfrproperty" (24). It carries the pyrB genes on theepisome (29) and integrates into the chromo-some of S. abony and also of S. typhimurium,S. paratyphi B, S. enteritidis (group D), S.

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senftenberg (group E,) and S. muenster (groupEl) to create Hfr behavior of the HfrHl type(22, 35; Makela, unpublished data). S. abonystrains carrying FS59 have the same orienta-tion of chromosome transfer as HfrH1 (Table7); FS59 in other species gives the same orien-tation of transfer but lower fertility. FS78 andFS79 were isolated in the same experiments asFS59; they appear similar to FS59.The FS403 factor, which carries the genes of

S. abony from the purG to ptsI region, wasisolated by Wyche et al. (Abstr. Annu. MeetingAmer. Soc. Microbiol., 1972).

E. coli F' factorsF' factors carrying E. coli chromosomal seg-

ments can be transmitted into Salmonella andhave been used for complementation analysis.In addition, they are of value for geneticmapping, for genes to be mapped in Salmonellaare often not in Hfr or multiply mutant F-strains. The strain carrying an unmapped mu-tant is infected with the F factor, usually anFlac + factor of E. coli which can be readilyselected in Lac- Salmonella strains; this strainis used as a donor with multiply auxotrophic F-strains (Table 2), and joint transfer of theunknown gene is tested.

DISCUSSIONThe behavior of F in Salmonella resembles

its behavior in E. coli K12. As seen in thisreport, F+ strains produce low-frequency chro-mosome mobilization, yielding about 10-5 to10- 6 recombinants per donor cell. The F+strains readily transfer F to other cells. Suchstrains carrying F are sensitive to the male-specific phages, though in some cases they donot show visible plaques (see Materials andMethods). A variety of strains with greatlyenhanced frequency of chromosome transfercan be isolated from these F+ strains. In S.typhimurium, such strains can be isolatedmost readily after prolonged storage of the F+strain, as described in Materials and Methods.In many cases, especially in S. typhimurium,these strains appear from the following evi-dence to be Hfr lines, defined in E. coli to resultfrom stable insertion of F into the chromosome.Firstly, in many cases these high-fertility lineshave a fixed and unique point of origin ofchromosome transfer, yielding up to 10-l re-combinants per donor cell, usually F-, forproximal genes. Secondly, selection for genestransferred at the distal extremity of the chro-mosome of a specific Hfr yields a compara-tively small number of recombinants, many of

which are Hfr strains which transfer the chro-mosome in the manner characteristic of the Hfrparent. Such recombinants have been detectedfrom crosses of SR305, SU354, and SA464, andpresumably result from transfer of the inte-grated F factor at the terminus of the chromo-some. Thirdly, curing of the high-fertilitystrain to the F- state, followed by reinfectionwith F in the few cases tested in S. typhimu-rium, does not usually lead to chromosometransfer from the recombinants as high as thatfrom the original Hfr line; such tests have beendone with SR305, SU418, and SU436.A high proportion of the high-fertility lines of

S. abony carry an infectious F and, when curedof F and reinfected, they regain their originalfertility. Such lines appear to have a sex-factor-affinity locus (sfa), postulated by Richter (27)to result from insertion of a portion of F into thechromosome, and resulting in a tendency of' Fto associate, more or less transiently, with thehomologous part of the chromosome, causingmobilization. Such lines are seen in S.typhimurium as well, for SR297 is obviously of'this type, as described above in the section onSR305. However, the SR305 (HfrA) lines whichcan be isolated from SR297, and which have 50to 100 times higher fertility, with the samepoint of origin as SR297, are postulated to bedue to a relatively stable insertion of F into thechromosome at the position of the sfa locus ofSR297.The interrupted conjugation experiments

with both S. typhimurium and S. abony Hfrstrains demonstrate that the Hfr behavior issimilar in these species to the well-describedevents in E. coli K12. There seem to be some,probably trivial, differences. Separation of themated pairs is not always easy to achievecompletely with equipment such as a Mini-mixer, and a Waring blendor may be necessary.The pairs that remain unseparated give rise toa variable background number of recombi-nants. This background increases with increas-ing time, partly reflecting a continuing processof pair formation. This is not the sole reason, asa similar picture was obtained when the matingmixture was diluted 1:100 after 5 min (5). Thelinear rise of the numbers of recombinants issharper and seems to occur earlier in matingsthat produce a higher final level of recombi-nants. This can be seen when the same wild-type gene is followed: thus entry times for prowith SH81 (HfrH1) can vary from 17 to 25 min,for gal from 30 to 40 min, with different S.abony recipients, and can be still longer with S.typhimurium recipients which give a very slug-gish rise of the curve. With an unstable Hfr, the

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rise of the curve may be slow and nonlinearbecause of continuing integration of F at the sfalocus. Yet these strains give a gradient ofrecombinants from proximal to distal as intypical Hfr strains. Mating Fig. 16, 17, and 18probably have a component of this sort. Thesefacts should lead to caution when deducingmap positions of unknown markers from pointsof entry. However, interrupted mating data arevery useful in deciding the order of genes, bycomparing their entry times in the same recipi-ent, or by determining linked transfer: theproportion of recombinants with a linkedmarker distal to the selected one is very lowamong the recombinants selected early, nearthe entry time of the selected marker, whereasthe proportion with a linked proximal marker issimilar among early and late recombinants.The comparisons of frequencies of recombi-

nants in homologous crosses (S. typhimuriumHfr x S. typhimurium F- or S. abony Hfr x S.abony F-) to those in the heterologous combi-nations (Tables 7, 8, 9) show that the homolo-gous crosses are on an average 10-fold moreproductive. This difference varies with differ-ent markers selected, and these differencesmay in some cases be due to induction ofprophages. These differences can be ascribed toreduced pair formation, host-controlled re-

striction, and lack of homology of the chromo-somes leading to impaired integration, but therelative importance of each component is un-

known. In order to transfer genes betweendifferent species, as in studies of the surfaceantigens or of determinants of pathogenicity, itis advisable to test Hfr donors of each speciesavailable as the outcome is unpredictable.FS59 has proven useful by giving chromosomemobilization from the same point of origin as

HfrHl when it is transferred into new strains.E. coli F' factors, such as F13 lac of E. coli, willmobilize the chromosome of Salmonella, butno specifilc point of origin is detectable.Though many Hfr strains of S. typhimurium

and S. abony are available, only a few F' factorsbearing segments of the Salmonella chromo-some have been reported. Most of these havebeen detected after interrupted mating of anHfr strain, followed by selection for a distallytransferred gene. In many laboratories, in-cluding ours, attempts have been made toisolate F' factors of Salmonella by using selec-tion for recombinants for genes transferredearly by the Hfr in crosses to recA recipientstrains (16). Both E. coli and S. typhimuriumrecipient strains have been used in suchcrosses, but for reasons not clear to us suchattempts have usually failed. However, Voll

(39) isolated FS400 his from a cross of S.typhimurium HfrK5 to an S. typhimuriumhis - recA mutant, with selection for the termi-nal marker, his+, following early interruptedmating, whereas Sanderson (unpublisheddata) failed with the same techniques, using arec+ recipient. This use of a recA recipient,even where the selection is for terminallytransferred genes, may be of general value.

Crosses of SA536, SA949, and SA977, threeHfr strains of S. typhimurium with selectionfor a distal gene, malA+, following 60 min ofmating yielded recombinants which are mostlydonor type for proximal genes, recipient typefor genes transferred in the intermediate part ofthe chromosome, only a few of which were Hfrand none F'. Crosses of SA970 and SA978, withselection for terminal genes, gave recombinantswhich were donor type for proximal genes,recipient type for intermediate genes, and fre-quently Hfr; however, F' strains were notdetected (Table 14). The detection of recombi-nants for proximal and distal genes, withouttransfer of the intermediate part of the chromo-some, suggests F' transfer, but the recombi-nants in no cases gave frequent transfer of theselected gene and therefore are assumed not tocarry F' factors.Many investigators have used F' factors of E.

coli, transferred into S. typhimurium, for test-ing complementation. Though these have thedisadvantage that one must assume a greatdegree of homology between the genetic mate-rial of the two genera in order to interpret theresults, such recombinants have the advantagethat there is little likelihood of recombinationbetween the genetic material of the chromo-some and of the episome.

ACKNOWLEDGMENTSWe acknowledge the assistance of C. Hall, R. McCann,

Marianne Hovi, Marjukka Brandt, and Seijasisko Suurna-kki.

Part of this investigation was supported by an operatinggrant from the National Research Council of Canada (NRCA 3684) and by joint grants from the National ScienceFoundation of the United States (GB7497) and the NationalResearch Council of Canada for the maintenance of aSalmonella Genetic Stock Centre at the University ofCalgary.

Part of the writing was done while KES held an Alexandervon Humboldt fellowship at the Max-Planck-Institut fur Im-munbiologie, Freiburg. The work in Helsinki was supportedby Sigrid Juselius Foundation and the Finnish Medical Re-search Council. The assistance of the editors and the re-viewers of this joumal in improving the manuscript is appre-ciated.

LITERATURE CITED1. Bagdian, G., and P. H. Mfikela. 1971. Antigenic

conversion by phage P27. I. Mapping of the

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prophage attachment site on the Salmonellachromosome. Virology 43:403-411.

2. Beckwith, J. R.. E. R. Signer, and W. Epstein.1966. Transposition of the lac region of' E. coli.Cold Spring Harbor Symp. Quant. Biol.31:39:3-401.

3. Cavalli-Sf'orza. L. L., and J. Lederberg. 1956.Isolation of' preadaptive mutants in bacteria bysib selection. Genetics 41:367- 381.

:3a. Curtiss, R., III, L. J. Charamella. D. R. Stal-lions, and J. A. Mavs. 1968. Parental t'unctionsduring conjugation in Escherichia coli K- 12.Bacteriol. Rev. 32:320-:348.

4. Davis, J. E.. J. H. Strauss, Jr.. and R. L.Sinsheimer. 1961. Bacteriophage MS2; anotherRNA phage. Science 134:1427.

5. De Haan, P. G., and J. D. Gross. 1962. Transt'erdelay and chromosomal withdrawal duringconjugation in Escherichia coli. Genet. Res.3:251-272.

6. Demerec. M., E. A. Adelberg, A. J. Clark. and P.E. Hartman. 1966. A proposal for a unit'ormnomenclature in bacterial genetics. Genetics54:61-76.

Edwards, P. R., and D. W. Bruner. 1942. Serolog-ical identif'ication ot' Salmonella cultures. Circ.Kentucky Agr. Exp. Sta. 54.

8. Glatzer. L.. D. A. Labrie, and F. B. Armstrong.1966. Transduction of' Salmonella typhimuri-um-Salmonella monteuideo hybrids. (enetics54:423-432.

9. Hayes. W. 1953. Observations on a transmissibleagent determining sexual diff'erentiation inBacterium co/i. J. Gen. Microbiol. 8:,2-88.

10. Hoi'schneider. P. H. 196:3. Untersuchungen fiber'kleine' E. co/i K12 Bacteriophagen. I. Mitt.Die Isolierung und Eigenschatten der 'klein-en" Bacteriophagen M12, M13, und M20. Z.Naturf'orsch. 186:20t3-205.

11. Jacob, F., and E. A. Adelberg. 1959. Transtert decharacteres genetiques par incorporat ion aut'acteur sexuel d'Escherichia co/i. C. R. Acad.Sci. 249:189-191.

12. Kuo. T. T., and B. A. D. Stocker. 1972. Mappingof rfa genes in Salmonella typhimurium byES18 and P22 transduction and by conjuga-tion. J. Bacteriol. 112:48-63.X

13. Lederberg, J., L. L. Cavalli, and E. M. Leder-berg. 1952. Sex compatibility in Escherichiacoli. Genetics 37:72)-730.

14. Levinthal. M., and R. D. Simoni. 1969. Geneticanalysis of carbohydrate transport-det'icientmutants of' Salmonella typhimurium. J. Bac-teriol. 97:250-255.

15. Low, B. 1965. Low recombination frequency formarkers very near the origin in conjugation inE. coli. Genet. Res. 6:469-473.

16. Low, B. 1968. Formation of' merodiploids inmatings with a class of rec recipient strains of'E. coli K12. Proc. Nat. Acad. Sci. U.S.A.60:160-167.

'7. Low, K. B. 1972. Hfr and F-prime strains of'Escherichia coli. Bacteriol. Rev. 36:587-607.

iS. T uderitz, O., A. M. Staub. and 0. Westphal.

N ET AL. BACTERIOL. REV.

1966. Immunochemistry of 0 and R antigens of'Salmonella and related Enterobacteriaceae.Bacteriol. Rev. 30:192-255.

19. Luderitz, O., 0. Westphal. A. M. Staub. and H.Nikaido. 1971. Isolation and chemical andimmunological characterization of' bacterial li-popolysaccharides. p. 145-2:33. In G. Wein-baum. S. Kadis. and S. J. Ajl (ed.), Microbialtoxins, vol. 4. Academic Press Inc., New York.

20. Miikelii, P. H. 1963. Hfr males in Salmonellaabonv. Genetics 48:423-429.

21. Makela, P. H., J. Lederberg, and E. M. Leder-berg. 1962. Patterns of sexual recombination inenteric bacteria. Genetics 47:1427-14.39.

22. Makela. P. H.. and 0. Makela. 1966. Salmonellaantigen 122: genetics of' f'orm variation. Ann.Med. Exp. Biol. Fenn. 44:310-:317.

23. MakelI, P. H., and B. A. D. Stocker. 1969. Ge-netics of polysaccharide biosynthesis. Annu.Rev. Genet. 3:291-32).

24. Makela. P. H., and L. Ziegler. 1966. An F'episome of' Salmonella abony. Acta Pathol.Microbiol. Scand. 67:547-554.

24a. Margolin, P. 196.3. Genetic f'ine structure of theleucine operon in Salmonella. Genetics48:441-457.

25. Matney. T. S.. and N. E. Achenbach. 1962. Acomment on the f'ertility of' F2 donor types of'Escherichia coli K12. Biochem. Biophvs. Res.Commun. 9:285-287.

26. Pittard, J., and E. M. Walker. 1967. Conjugationin Escherichia coli: recombination events interminal regions of' transferred deoxyribonu-cleic acid. J. Bacteriol. 94:1656-166.3.

27. Richter. A. 1961. Attachment of' wild-type Ff'actor to a specif'ic chromosomal region in avariant strain of' Escherichia coli K12: thephenomenon ot' episomic alternation. Genet.Res. 2:333-345.

28. Sanderson. K. E. 1965. Information transfer inSalmonella typhimurium. Proc. Nat. Acad.Sci. U.S.A. 53:1:3,35-1:340.

29. Sanderson. K. E. 197t). Current linkage map ofSalmonella typhimurium. Bacteriol. Rev.34:176- 193.

.30. Sanderson. K. E. 1972. Linkage map of Sal-monella typhimurium. edition IV. Bacteriol.Rev. 36:558-586.

:31. Sanderson, K. E.. and M. Demerec. 1965. Thelinkage map ot' Salmonella typhimurium. Gen-etics 51:897-91.3.

32. Sanderson, K. E.. and C. A. Hall. 1970. F-primef'actors of' Salmonella typhimurium and aninversion between S. tvphimurium and E. coli.Genetics 64:215-228.

33. Sanderson, K. E., and H. Saeed. 1972. Insertionof' the F tactor into the cluster ot' rfa (rough A)genes of Salmonella typhimurium. J. Bacteriol.112:64-73.

34. Sanderson, K. E., and Y. A. Saeed. 1972. P22-mediated transduction analysis ot' the rough A(rfa) region ot' the chromosome ot' Salmonellatyphimurium. J. Bacteriol. 112:58-6,3.

35. Sarvas. M. 1967. Inheritance of' Salmonella Ti

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aptigen. Ann. Med. Exp. Biol. Fenn.45:447-471,

36. Stocker, B. A. D., and P. H. Makela. 1971.Genetic aspects of biosynthesis and structureof Salmonella lipopolysaccharide, p. 369-438.In G. Weinbaum, S. Kadis, and S. J. Ajil (ed.),Microbial toxins, vol. 4. Academic Press Inc.,New York.

37. St. Pierre, M. L. 1968. Isolation and mapping ofSalmonella typhimurium mutants defective inthe utilization of trehalose. J. Bacteriol.95:1185-1186.

38. Taylor, A. L., and C. D. Trotter. 1972. Linkagemap of Escherichia coli strain K12. Bacteriol.Rev. 36:504-524.

39. Voll, M. J. 1972. Derivation of an F-merogenoteand a 080 high-frequency transducing phage

carrying the histidine operon of Salmonella. J.Bacteriol. 109:741-750.

40. Watanabe, T., T. Arai, and T. Hattori. 1970.Effects of cell wall polysaccharide on the mat-ing ability of Salmonella typhimurium. Nature(London) 225:70-71.

41. Wiedemann, B., and G. Schmidt. 1971. Struc-ture and recipient ability in E. coli mutants.Ann. N.Y. Acad. Sci. 182:123-125.

42. Wilkinson, R. G., P. Gemski, Jr., and B. A. D.Stocker. 1972. Non-smooth mutants of Sal-monella typhimurium: differentiation byphage sensitivity and genetic mapping. J. Gen.Microbiol.

43. Zinder, N. D. 1960. Sexuality and mating inSalmonella. Science 131:924-926.

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