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groups (A, B, C, and D; now called asg, bsg, csg, and dsg for a signaling, etc.) are recognized in the initial set of 50 mutants. There appear to be at least four different signals. Each of the four types of Hagen mutants has a different developmental phenotype and corresponds to a different locus or set of loci. 26 Acknowledgments Work on motility from the author's laboratory was supported by a grant from the National Science Foundation DCB-8903705 and on cell-to-cell signaling by Public Health Service grant GM23441 from the Institute for General Medical Sciences.
[17] G e n e t i c s o f C a u l o b a c t e r crescentus
By BERT ELY Introduction
Caulobacter crescentus is a gram-negative, aquatic bacterium which is characterized by an unusual cell cycle (for a recent review, see Newton~). Each cell division results in two different cell types, a sessile stalked cell and a motile swarmer cell. The stalked cell immediately begins to replicate its chromosome and prepare for the next ceil division. Midway through chromosome replication, differentiation begins at the pole opposite the stalk, resulting in the synthesis of a flagellum, pill, and membrane phage receptors. This differentiated pole confers motility on the progeny swarmer cell when cell division occurs. The swarmer cell is an immature cell which must go through a maturation process before it can replicate its chromosome and divide. After a period of motility, the polar flagellum is lost, and a stalk is synthesized in its place. This new stalked cell then is capable of DNA replication and cell division. Thus, each cell cycle includes two times when differentiation occurs, the transition from a swarmer to a stalked cell and the differentiation of the flagellar pole of the predivisional cell. Furthermore, since each cell division results in two different cell types, differential segregation of cell components occurs. This differential segregation is most obvious at the poles of the cell but occurs with soluble components as well. Thus, the Cauiobacter cell cycle has features which are normally found only in more complex organisms. I A. Newton, in "Bacterial Diversity" (K. F. Chater and D. A. Hopwood, eds.), Vol. 2, p. 199. Academic Press, New York, 1989.
METHODS IN ENZYMOLOGY,VOL. 204
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Since a well-developed genetic system has become available for C. crescentus, it is a premier organism for the study of the spatial distribution of gene products and the cell cycle control of gene expression. 2
Growth of Caulobacter crescentus
Caulobacter crescentus lives in ponds and streams and is a scavenger in nature. It is adapted to growth in low nutrient conditions and grows poorly, if at all, in media designed for Escherichia coli. We grow C. crescentus with vigorous aeration at 33° in either a complex medium (PYE) or one of two defined media (M2 or PIG). PYE is a modification of the medium described by Poindexter ~ and consists of 0.2% peptone (Difco, Detroit, MI), 0.1% yeast extract (Difco, Detroit, MI), 0.8 mM MgSO4, and 0.5 mM CaC12.4 The addition of calcium is necessary since there is not always enough present in the other components to ensure optimal growth. The defined medium 4 M2 is prepared from a 10 x concentrated base consisting of 17.4 g Na2HPO 4 , 10.6 g KH2PO 4 , and 5 g NH4CI per liter of high-quality distilled water. (We use an Ultrascience Hi-Q glass still. Water prepared in conventional distillation systems contains volatile organics which inhibit the growth of C. crescentus, resulting in doubling times of 8-10 hr. Normal doubling times are 2-3 hr in defined medium prepared with high-quality water.) To prepare M2 medium, the 10 × M2 stock is diluted and autoclaved. After cooling, I0 ml each of 30% (w/v) glucose, 50 mM MgCI2, 50 mM CaC12, and 1 mM FeSO4 in 0.8 mM EDTA, pH 6.8, are added aseptically to each liter of medium. For solid medium, equal volumes of 2 x M2 and 2% agar are combined after autoclaving, and the CaC12 is omitted as it is provided by the agar. Some strains with mutations causing pleiotropic effects do not grow well in M2 medium owing to the high phosphate levels. Therefore, a low phosphate medium, PIG is adopted) The base for PIG medium is l0 × I and consists of 5 g NH4CI and 6.8 g imidazole with the pH adjusted to 7.0 with HCI. PIG solid medium is made in the same fashion as M2 medium except that 10 ml of 0.1 M sodium glutamate, pH 7, and 1 ml of 0.1 M sodium phosphate buffer, pH 7, are added in addition to the other supplements. Liquid medium consists of 1 × I plus 20 ml Hutner's mineral
2 B. Ely and L. Shapiro, Genetics 12,3, 427 (1989). 3 j. S. Poindexter, Bacteriol. Rev. 28, 231 (1964). 4 R. C. Johnson and B. Ely, Genetics 86, 25 (1977). 5 p. V. Schoenlein, L. M. Gallman, and B. Ely, J. Bacteriol. 171, 1544 (1989).
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base, 6 I0 ml of 30% glucose, 10 ml of 0.1 M sodium phosphate buffer, pH 7, and I0 ml of 0.1 M sodium glutamate, pH 7, per liter. Storage of Genetic Stocks For long-term studies, it is important to maintain viable genetic stocks in an unchanged state. We have found that C. c r e s c e n t u s can be maintained at - 7 0 ° indefinitely without loss of viability. Cultures of strains to be stored are grown to stationary phase in PYE broth, and 1-ml aliquots are added to sterile vials containing 2 drops of dimethyl sulfoxide (DMSO). Adhesive paper labels are attached to the caps and sealed with clear fingernail polish to provide protection against moisture, and the vials are transferred to a - 7 0 ° freezer. The dimethyl sulfoxide promotes uniform freezing and prevents the formation of large ice crystals which could disrupt cell membranes. Strains are retrieved by scraping the surface of the frozen stock with a sterile stick and using the stick to inoculate a petri dish or culture tube. In this manner, the same stocks can be used repeatedly without thawing. We have found that stocks prepared in this manner maintain close to 100% viability for at least 15 years. It is important that cultures for frozen stocks be grown to stationary phase since frozen stocks made from log-phase cultures begin losing viability within the first year of storage. Mutant Isolation Many kinds ofC. c r e s c e n t u s mutants can be obtained without mutagen¢sis. Antibiotic-resistant mutants can be obtained by direct selection, and motility mutants can be obtained by serial enrichment procedures for nonmotile cells on soft afar plates. 7 Auxotrophic mutants can be obtained after enrichment for nongrowing cells. Since wild-type C a u l o b a c t e r is resistant to penicillin, however, fosfom~cin or D-cycloserine must be used instead. 4 The standard procedures described for penicillin enrichment are used for either of the two antibiotics, and prior mutagenesis is unnecessary. Cultures are grown in PYE, centrifuged, and resuspended in defined medium. After one doubling has occurred in defined medium, either 100 ~g/ ml D-cycloserine or 500/.~g/ml fosfomycin is added. Samples arc removed 13, 15, and 18 hr after the addition of antibiotic, spun, and resuspended to remove the antibiotic and plated on PYE medium. The resulting colonies are replicated onto defined medium to identify those with nutritional requirements. Auxotrophs are 5 to 10% of the survivors in typical experiG. Cohen-Bazire, W. R. Sistrom, and R. Y. Stanier, J. Cell. Comp. Physiol. 49, 25 (1957). 7 R. C. Johnson and B. Ely, J. Bacteriol. 137, 627 (1979).
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ments. Auxotrophs and motility mutants can also be obtained by transposon mutagenesis (see below). Generalized Transduction
Generalized transduction involves the use of a bacteriophage to transfer genetic markers from one strain to another. For C. crescentus the bacteriophage tbCR30 is excellent for this purpose since it can transfer large pieces of DNA and is easy to g r o w ) The basic strategy is to grow tbCR30 on the donor strain to allow the random mispackaging of fragments of the donor DNA into bacteriophage particles. The resulting lysate is irradiated with ultraviolet light to inactivate the viral genomes and then stored over chloroform to kill any residual bacteria. Transduction experiments are performed by mixing a dilution of the irradiated donor lysate with the recipient strain on the surface of a petri dish containing a selective medium. Colonies which grow should contain the selected marker from the donor.
Preparation of Transducing Lysates An overnight culture of the strain chosen to be a donor is prepared, and 0.2 ml of this culture is mixed with 0.1 ml of a 10-4 dilution of ~bCR30 lysate (unirradiated) in 3 ml of melted PYE soft agar. The mixture is immediately poured onto the surface of a fresh PYE plate and allowed to harden. After overnight incubation at 33° , overlapping plaques should be observed. If the lysis appears satisfactory, 5 ml of PYE broth is pipetted onto the petri dish, which is refrigerated overnight to allow the bacteriophage to diffuse out of the agar into the liquid overlay. The next day, the broth is decanted into an empty 60-ram-diameter petri dish. The cover is removed and the contents are swirled under a germicidal ultraviolet light at a distance of approximately 45 cm for 5 min. (If the petri dish is swirled by hand, protective gloves should be worn.) After irradiation, the contents of the dish are poured into a glass screwcapped tube, a few drops of chloroform are added, and the tube is shaken vigorously to ensure that all parts of the tube come in contact with the chloroform. A drop of this lysate is placed on a PYE plate and incubated to verify that all of the contaminating bacteria have been destroyed. A good lysate should have a titer of more than 101°plaque-forming units (pfu) per milliliter before irradiation and less than 105 pfu/ml after irradiation. Although loss of transducing ability occurs slowly at 4 °, phage lysates can be refrigerated for several years and still maintain good donor ability. 8 B. Ely and R. C. Johnson, Genetics 87, 391 (1977).
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Transduction
Transduction experiments are performed by mixing 0.1 ml of a fresh overnight culture of the recipient strain with 0.1 ml of a 10 -2 or 10 -3 dilution of the donor lysate. The mixing is done on the surface of a petri dish containing the appropriate selective medium. Colonies appear after incubation for 2 to 5 days at 33 °. In the case where tetracycline resistance is used as a selective marker, a 10 ml overlay of melted PYE agar is poured on top o f a PYE plate containing I mg/ml tetracycline, and the plate is used as soon as the overlay hardens. The overlay allows time for expression of tetracycline resistance before sufficient tetracycline comes in contact with the recipient cells to cause cell death. When transduction experiments are performed on minimal medium, ~bCR30 growth is inhibited and phageresistant transductants are rarely observed. When transduction experiments are performed on a PYE medium, however, phage replication occurs, and a significant proportion of the transductant colonies will have mutated to phage resistance. In either case, 102 to 104 transductants are obtained per plate depending on the titer of the original donor phage lysate and the recipient marker being transduced. Conjugation Conjugation involves the transfer of DNA from one cell to another through direct cell contact. In the case of C. crescentus, this transfer can be mediated by the conjugative plasmid R P 4 . 9 RP4 is a broad host range plasmid which can be transferred among many gram-negative bacteria and will mediate intraspecies chromosomal transfer. Donor strains containing the RP4 plasmid produce pili which establish contact with the recipient strain and provide a conduit for the actual transfer of DNA from the donor to the recipient. Plasmid Transfer Plasmid transfer is an efficient process which can be accomplished by simply allowing contact between the donor and recipient strains. The simplest method is the cross-streak method. 1° A selective medium is chosen which will inhibit growth of both the donor and the recipient strains but will allow growth of those recipient cells which obtain the plasmid from the donor. Overnight cultures of both strains are prepared, and using a 0.1-ml pipette, approximately 20/.d of the donor culture is streaked 9 B. Ely, Genetics 91, 371 (1979). 1o B. Ely, Genetics 78, 593 (1974).
[17]
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377
across the surface of a petri dish containing a selective medium. The culture is drawn into the pipette by capillary action, and the pipette is held like a pencil to draw a line of culture fluid across the plate. After the fluid in the donor streak has soaked into the medium, streaks of one or more recipient strains are made in a similar manner, perpendicular to the donor streak. As the pipette containing the recipient culture crosses the donor streak, donor cells are dragged along and mixed with the recipient cells. After incubation for 2 days, growth is observed throughout the half of the recipient streak where mixing of the two strains occurred. The other half of the recipient streak and all but the intersection of the donor streak serve as controls to monitor for spontaneous mutation or contamination. Plasmid-containing recipient strains are purified by streaking for single colonies on selective medium. Chromosome Transfer
Chromosome transfer is less efficient than plasmid transfer and requires a longer time for the actual transfer process to occur. To maximize the efficiency of transfer, the donor and recipient strains are held together on a solid support while the transfer occurs. The filter mating procedure 9 is accomplished by mixing 0.1 ml of an overnight culture of the donor with 0.5 ml of an overnight culture of the recipient, then filtering the mixture through a sterile 2.5-cm-diameter 0.45-txm filter. After the excess liquid is removed, the filter is placed on a fresh PYE plate and incubated at 33° for 2 to 3 hr. The filter is then placed in a tube containing 1 ml of PYE broth and agitated vigorously to resuspend the cells. Aliquots (0.2 ml) of the resuspended cells are spread on the surface of petri dishes containing a medium which is selective for both the donor and the recipient cells. Generally 50 to 200 colonies appear after 3 to 5 days at 33 °. Transfer of up to one-sixth of the C. crescentus chromosome can be detected with this procedure.
Transposon Mutagenesis Transposition of both Tn5 and Tn7 occurs in C. crescentus. H However, Tn7 inserts at a single site on the chromosome and, therefore, is of limited usefulness for genetic studies.12 O n the other hand, Tn5 and its derivatives appear to transpose randomly and provide a means for obtaining a selectable marker in or near any C. crescentus gene. 11 B. Ely and R. H. Croft, J. Bacteriol. 149, 620 (1982). l: B. Ely, J. Bacteriol. 151, 1056 (1982).
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Isolation o f New Transposon Insertions
Transposon mutagenesis occurs after the transposon is introduced into target strain on a vector which is unstable in C. crescentus. ~1 P-type plasmids containing bacteriophage M u 13 o r based on a ColE1 replicon TM provide excellent vehicles for the transient insertion of a transposon. Overnight cultures of the recipient strain and an E. coli strain containing the donor plasmid are prepared, and the filter mating procedure is carried out as described for conjugation experiments. Approximately 30 to 100 antibiotic-resistant colonies are obtained per plate, and each represents an independent transposition event. If a plasmid containing the Tn5 derivative Tn5-VB32 is used, 15promoter fusions can be obtained. Tn5-VB32 contains a promoterless neomycin phosphotransferase II (NPTase II) gene near one end which can be expressed from an adjacent promoter. 16If Tn5-VB32 is inserted into a C. crescentus gene in the proper orientation, the NPTase II gene is expressed from the promoter of that gene and serves as a reporter to monitor the expression of the mutated gene.17
Identification of Transposon Insertions Linked to Specific Markers
To obtain transposon insertions linked to a specific marker, the random mutagenesis procedure is used to generate approximately 500 colonies containing independent insertions. The resulting colonies are mixed together and suspended in PYE broth. An aliquot of the resulting suspension is then used as the host for the preparation of a ~bCR30 lysate as described above. Transduction experiments can be performed using the resulting lysate with selection for the antibiotic resistance encoded by the transposon. Subsequent screening for the loss of a recipient marker results in the identification of transposon insertions which are linked to the marker of interest. Phage lysates can then be prepared from individual transposon mutants, and transduction experiments can be performed to determine the proximity (linkage) of the transposon to the gene interest.
13j. E. Behringer, J. L. Brynon, A. V. Buchanon-Wallaston, and A. W. B. Johnston, Nature (London) 276, 663 (1978). 14 B. Ely, Mol. Gen. Genet. 200, 302 (1985). t5 V. Bellafatto, L. Shapiro, and D. A. Hodgson, Proc. Natl. Acad. Sci. U.S.A. 81, 1035 (1984). 16R. Champer, R. Bryan, S. L. Gomes, M. Purucker, and L. Shapiro, Cold Spring Harbor Syrup. Quant. Biol. 50, 831 (1985). 17 R. Champer, A. Dingwall, and L. Shapiro, J. Mol. Biol. 194, 71 (1987).
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Exchange of Transposon-Encoded Antibiotics Resistance Sometimes it is useful to change the antibiotic resistance encoded by a specific transposon insertion. TM The kanamycin resistance encoded by Tn5 can be exchanged for tetracycline resistance using pBEE132 to introduce the Trd-132 into the Trt5-containing strain. Transfer of pBEE132 is accomplished by the filter mating procedure described above. Colonies which are tetracycline resistant and kanamycin sensitive have the tetracycline resistance of Tn5-132 substituted for the kanamycin resistance of the inserted Tn5 element. Electroporation Electroporation involves the use of a brief electric shock to depolarize the cell membrane and allow the uptake of DNA. Electroporation works well with C. crescentus for the uptake of plasmid DNA using procedures developed for E. coli. 19 However, in contrast to the case for E. coli, experiments with cut and religated plasmid DNA have not been successful. To prepare cells for electroporation, a 500-ml culture is grown at 33° to mid-log phase in PYE. The culture is divided into two centrifuge bottles and spun at 6000 g for 5 min. The pellets are resuspended in 100 ml distilled water, spun at 6000 g for 5 rain, and then resuspended in 100 ml of distilled water a second time and spun as before. The washed pellets are resuspended in l0 ml of 10% glycerol, combined and transferred to a 50ml centrifuge tube, spun at 3000 g for 5 rain, and resuspended in 2 ml of 10% glycerol. The concentrated cells are divided into 200-/.d aliquots and frozen at - 7 0 ° . For electroporation, a tube is thawed, and 40/zl of cells is placed in a microfuge tube along with 0.1 to 1/~g of DNA in a small volume of distilled water (or very dilute buffer). After 1 rain on ice, the mixture is transferred to an ice-cold Bio-Rad (Richmond, CA) Gene Pulser cuvette with a 0.2cm electrode gap and subjected to a 2.5-kV shock with the capacitor set at 25/~F (microfarad) and the resistance on the pulse controller set at 200 ohms. Under these conditions, the time constant is 5 msec. After this treatment, the contents of the cuvette are transferred to a culture tube containing 1 ml of PYE, and the cells are incubated at 33 ° with aeration for 1 hr. After the recovery period, the cells are plated on the appropriate selective medium. We routinely obtain 104 to 105 transformants per microgram of DNA. 18 B. Ely and T. Ely, Genetics 123, 649 (1989). 19 A. K. Ellgaard, D. A. Mullin, and S. A. Minnich, Abstr. Annu. Meeting Am. Soc. Microbiol., p. 208 (1989).
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Pulsed-Field Gel Electrophoresis Pulsed-field gel electrophoresis (PFGE) allows one to separate very large DNA fragments. A genomic map of the C. crescentus chromosome has been generated by aligning DraI, AseI, and SpeI restriction maps with the genetic map. ~s Therefore, the map location of transposon insertions can be determined by PFGE. This procedure is approximately 100 times faster than using a combination of conjugation and transduction to determine map locations. 2° A current version of the genomic map is shown in Fig. 1.
Preparation o f DNA Plugs DNA plugs are prepared by a modification of the method of Smith and Cantor. 2~A 2.5-ml aliquot of an overnight culture of a C. crescentus strain is spun at 4000 g in a refrigerated centrifuge, and the resulting pellet is resuspended in 2 ml of 10 mM Tris, pH 7.6, containing 1 M NaCI. The mixture is spun a second time, resuspended in 1 ml of the same buffer, and heated to 37 °. One-half of the suspension is then mixed with an equal volume of melted I% (w/v) agarose (PFG grade) previously equilibrated to 65 ° . The mixture is immediately transferred to plug molds and allow to cool. After cooling, each set of plugs is removed from the mold and transferred to a disposable plastic test tube (15 × 100 mm) which contains 1.8 ml of 1% (w/v) sarkosyl in 0.5 M E D T A (pH 9-9.4) and 0.2 ml of 10 nag/ ml proteinase K. After overnight incubation at 50°, the liquid is removed by aspiration and replaced with 2 ml of 10 mM Tris, pH 7.5, 1 mM EDTA and 30 tzl of a phenylmethylsulfonyl fluoride (PMSF) stock (17.4 mg/ml in 100% ethanol). After 1 hr on a rotator at room temperature, the liquid is removed by aspiration and replaced by an identical mixture. After a second hour, the procedure is repeated 3 times without the PMSF. After the final wash, the tube containing the plugs is refrigerated until needed. We have found that DNA-containing plugs can be stored in the refrigerator for several years.
Pulsed-Field Gel Electrophoresis Techniques PFGE experiments are carried out by digesting DNA-containing agarose plugs with restriction enzymes which cut the genome infrequently. The resulting DNA fragments are then resolved by insertion of the plug into an agarose gel and subjecting the DNA to PFGE. Restriction digests are carried out by placing a small segment of a DNA-containing agarose 20 A. Dingwall, L. Shapiro, and B. Ely, Methods 1, 160 (1990). 21 C. L. Smith and C. R. Cantor, Cold Spring Harbor Syrup. Quant. Biol. 51, 115 (1986).
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381
4000
trpE aroF I
l~c cysE
thi eux
metD 3500 hisB I
~10
500
-gO ' 0- ' ~ ~ c y s B
hisD
3000 aroG
t000 hunA pilA
aroC ,/ serA
3O5 hung trpB
,
gltA
~)/ I 15oo proA leuA
cysA hisG 2000
FIG. 1. Caulobacter c r e s c e n t u s genonlic map. Inner Circles show fragmentsgenerated by DraI, AseI, and SpeI restrictiondigests, respectively,with SpeI fragmentsrepresented on the innermostcircle. Fragment sizes are indicatedin kilobases. plug in a microfuge tube along with approximately 10 units of restriction enzyme, 10/.d of 10 x restriction buffer, and 50/zl distilled water. After incubation at 37° for 1 hr, the plug fragments are removed with a spatula and inserted directly into the gel wells for PFGE. Table I shows conditions which give good resolution of various size ranges of DNA fragments. After electrophoresis, the gel is stained with ethidium bromide and photographed. DNA fragments containing a Tn5 insertion can usually be identified by the altered migration of the corresponding band on the gel. Differences as small as 2% of the size of the fragment can be resolved under optimal conditions. Alternatively, the DNA can be blotted to nylon membranes and bands identified by hybridization.
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TABLE I CONDITIONS FOR SEPARATING VARIOUS SIZE RANGES OF DNA FRAGMENTS BY PULSED-FIELD GEL ELECTROPHORESIS Fragment size range (kb)
Pulse time (sec)
Voltage (V)
Time (hr)
5-50 20-100 50-250 100-400 200-1000
1 3 10 20 45
200 200 200 200 200
10 10 14 14 16
Complementation Complementation experiments involve the transfer of cloned DNA into a mutant strain to determine if the transferred DNA can correct the mutant phenotype. Positive results can be obtained as a result of complementation, recombination, or reversion. False positives arising from reversion are detected by the use of appropriate controls, and recombination can be prevented by the use of a recipient strain which is rec-.22 Construction o f rec- Strains Strains defective in recombination are constructed by use of strain PC7070 for cotransduction of the recA526 allele with a linked Tn5 insertion. 23Phage grown on PC7070 is used to transduce a kanamycin-sensitive strain containing the mutation to be complemented. The presence of the recA526 allele is demonstrated by an increased sensitivity to irradiation with ultraviolet light. Strains containing the mutation are sensitive to a 30sec exposure to a germicidal lamp at a distance of 45 cm, whereas those containing the wild-type allele are resistant to this exposure. In practice, a master plate of transductant colonies is replicated onto two plates. One of the replicas is exposed to the ultraviolet light, and both are incubated overnight. Colonies which grow only on the unirradiated plate contain the rec allele. Alternatively, the recA allele can be transferred by cotransduction with the cysB gene (2% linkage) in order to avoid the use of a Tn5 insertion (A. Newton, personal communication, 1978). 22 E. A. O'Neill, R. H. Hynes, and R. A. Bender, Mol. Gen. Genet. 198, 275 (1985). 23 N. Ohta, M. Masurekar, D. A. Mullin, and A. Newton, J. Bacteriol. 172, 7027 (1990).
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Complementation o f rec- Strains
Complementation experiments are performed using the cross-streak method described earlier. Complementation of a nutritional mutation can be observed directly by performing the transfer on defined medium. Complementation of motility mutations or other mutations where direct selection is not possible is done by using antibiotic resistance to select for transfer of the plasmid containing the cloned DNA. The plasmid-containing strain is then purified and checked for the loss of the mutant phenotype. Positive complementation results can be verified by growing the strain under nonselective conditions and screening for loss of the plasmid. The resulting strains are then tested for the reappearance of the mutant phenotype. Vectors based on RSF1010 are lost very quickly in C. crescentus, so few colonies will contain the plasmid after plating an aliquot of a culture grown overnight on PYE broth. In contrast, when the same procedure is used with vectors based on pRK290, approximately 1% of the resulting colonies have lost the plasmid, so a greater number of colonies need to be screened to obtain the desired segregants. Inactivation of Chromosomal Genes Chromosomal genes can be inactivated, or specific mutations can be introduced into the chromosome, through the use of cloned genes. The desired mutation is introduced into a cloned copy of the gene, and the plasmid containing the mutated gene is transferred to the appropriate C. creseentus strain. Recombination between the chromosomal and the plasmid copies of the gene usually results in the integration of the plasmid into the chromosome. Thus, the resulting strain will have both the wildtype and the mutated forms of the gene. Subsequent loss of the plasmid antibiotic resistance owing to a second recombination event results in the loss of one copy of the gene. Depending on where recombination occurs these segregants will have either the wild-type or the mutated version of the gene. Vectors for gene replacement should be incapable of replication in C. crescentus. Two types of vectors are appropriate. The first are vectors which contain the sequences necessary for mobilization by the IncP transfer system. 24These plasmids replicate well in E. coil and can be transferred from E. coil to C. crescentus at a high frequency 25(also B. Ely, unpublished observations, 1987). However, they cannot be replicated in C. crescentus, so they are lost unless recombination occurs to form a cointegrate. Trans24 R. Simon, U. Pfiefer, and A. Puhler, Bio Technology 1, 784 (1983). S. A. Minnich, N. Ohta, N. Taylor, and A. Newton, J. Bacteriol. 170, 3953 (1988).
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duction can then be used to obtain true gene replacements where the resulting strain contains a single copy of the mutated gene. 25A transducing lysate is prepared on a pool of cointegrate colonies and used to transduce the appropriate C. crescentus strain with selection for a marker either within the gene of interest or closely linked to it. The resulting colonies are then screened for the presence of the mutated gene and the absence of the plasmid antibiotic resistance. This procedure can also be used to transfer genes to a new chromosomal location using homology between Tn5 insertions for recombination (N. Ohta, L. S. Chen, D. A. Mullen, and A. Newton, personal communication). In this case, a plasmid containing a Tn5 element will integrate at the site of any chromosomal Tn5 insertion. A second procedure for the isolation of gene replacements is the use of electroporation to introduce the plasmid containing the mutated gene into the appropriate C. crescentus strain (J. Malakooti and B. Ely, unpublished observations, 1990). In this case, any vector can be used as long as it does not replicate in C. crescentus. After the plasmid containing the mutated gene is constructed in E. coli, plasmid DNA is isolated and used to transform the C. crescentus strain with selection for the plasmid antibiotic resistance. If ampicillin is used as the selective marker, a derivative of the ampicillin-sensitive strain SCl107 must be used as the host. The resulting colonies will be cointegrates, and gene replacements can be isolated as described above. Cointegrates are obtained at a frequency of 103 per microgram of plasmid DNA. This procedure also can be used to introduce a new restriction site into the genome at a particular location. Homologous recombination between a cloned fragment in the appropriate vector and the chromosome would result in the integration of the plasmid. If the plasmid contains one of the restriction sites used for PFGE mapping, the position of the cloned DNA could be readily determined as described above.
[18] G e n e t i c A n a l y s i s o f Agrobacteriurn
By GERARD A. CANGELOSI, ELAINE A. BEST, GLADYS MARTINETTI, and EUGENE W. NESTER
Introduction Genetic infection by Agrobacterium species is the only verified example of natural genetic exchange between the prokaryotic and eukaryotic kingdoms. Infection of plants by Agrobacterium strains containing tumorMETHODS IN ENZYMOLOGY,VOL. 204
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groups (A, B, C, and D; now called asg, bsg, csg, and dsg for a signaling, etc.) are recognized in the initial set of 50 mutants. There appear to be at least four different signals. Each of the four types of Hagen mutants has a different developmental phenotype and corresponds to a different locus or set of loci. 26 Acknowledgments Work on motility from the author's laboratory was supported by a grant from the National Science Foundation DCB-8903705 and on cell-to-cell signaling by Public Health Service grant GM23441 from the Institute for General Medical Sciences.
[17] G e n e t i c s o f C a u l o b a c t e r crescentus
By BERT ELY Introduction
Caulobacter crescentus is a gram-negative, aquatic bacterium which is characterized by an unusual cell cycle (for a recent review, see Newton~). Each cell division results in two different cell types, a sessile stalked cell and a motile swarmer cell. The stalked cell immediately begins to replicate its chromosome and prepare for the next ceil division. Midway through chromosome replication, differentiation begins at the pole opposite the stalk, resulting in the synthesis of a flagellum, pill, and membrane phage receptors. This differentiated pole confers motility on the progeny swarmer cell when cell division occurs. The swarmer cell is an immature cell which must go through a maturation process before it can replicate its chromosome and divide. After a period of motility, the polar flagellum is lost, and a stalk is synthesized in its place. This new stalked cell then is capable of DNA replication and cell division. Thus, each cell cycle includes two times when differentiation occurs, the transition from a swarmer to a stalked cell and the differentiation of the flagellar pole of the predivisional cell. Furthermore, since each cell division results in two different cell types, differential segregation of cell components occurs. This differential segregation is most obvious at the poles of the cell but occurs with soluble components as well. Thus, the Cauiobacter cell cycle has features which are normally found only in more complex organisms. I A. Newton, in "Bacterial Diversity" (K. F. Chater and D. A. Hopwood, eds.), Vol. 2, p. 199. Academic Press, New York, 1989.
METHODS IN ENZYMOLOGY,VOL. 204
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Since a well-developed genetic system has become available for C. crescentus, it is a premier organism for the study of the spatial distribution of gene products and the cell cycle control of gene expression. 2
Growth of Caulobacter crescentus
Caulobacter crescentus lives in ponds and streams and is a scavenger in nature. It is adapted to growth in low nutrient conditions and grows poorly, if at all, in media designed for Escherichia coli. We grow C. crescentus with vigorous aeration at 33° in either a complex medium (PYE) or one of two defined media (M2 or PIG). PYE is a modification of the medium described by Poindexter ~ and consists of 0.2% peptone (Difco, Detroit, MI), 0.1% yeast extract (Difco, Detroit, MI), 0.8 mM MgSO4, and 0.5 mM CaC12.4 The addition of calcium is necessary since there is not always enough present in the other components to ensure optimal growth. The defined medium 4 M2 is prepared from a 10 x concentrated base consisting of 17.4 g Na2HPO 4 , 10.6 g KH2PO 4 , and 5 g NH4CI per liter of high-quality distilled water. (We use an Ultrascience Hi-Q glass still. Water prepared in conventional distillation systems contains volatile organics which inhibit the growth of C. crescentus, resulting in doubling times of 8-10 hr. Normal doubling times are 2-3 hr in defined medium prepared with high-quality water.) To prepare M2 medium, the 10 × M2 stock is diluted and autoclaved. After cooling, I0 ml each of 30% (w/v) glucose, 50 mM MgCI2, 50 mM CaC12, and 1 mM FeSO4 in 0.8 mM EDTA, pH 6.8, are added aseptically to each liter of medium. For solid medium, equal volumes of 2 x M2 and 2% agar are combined after autoclaving, and the CaC12 is omitted as it is provided by the agar. Some strains with mutations causing pleiotropic effects do not grow well in M2 medium owing to the high phosphate levels. Therefore, a low phosphate medium, PIG is adopted) The base for PIG medium is l0 × I and consists of 5 g NH4CI and 6.8 g imidazole with the pH adjusted to 7.0 with HCI. PIG solid medium is made in the same fashion as M2 medium except that 10 ml of 0.1 M sodium glutamate, pH 7, and 1 ml of 0.1 M sodium phosphate buffer, pH 7, are added in addition to the other supplements. Liquid medium consists of 1 × I plus 20 ml Hutner's mineral
2 B. Ely and L. Shapiro, Genetics 12,3, 427 (1989). 3 j. S. Poindexter, Bacteriol. Rev. 28, 231 (1964). 4 R. C. Johnson and B. Ely, Genetics 86, 25 (1977). 5 p. V. Schoenlein, L. M. Gallman, and B. Ely, J. Bacteriol. 171, 1544 (1989).
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base, 6 I0 ml of 30% glucose, 10 ml of 0.1 M sodium phosphate buffer, pH 7, and I0 ml of 0.1 M sodium glutamate, pH 7, per liter. Storage of Genetic Stocks For long-term studies, it is important to maintain viable genetic stocks in an unchanged state. We have found that C. c r e s c e n t u s can be maintained at - 7 0 ° indefinitely without loss of viability. Cultures of strains to be stored are grown to stationary phase in PYE broth, and 1-ml aliquots are added to sterile vials containing 2 drops of dimethyl sulfoxide (DMSO). Adhesive paper labels are attached to the caps and sealed with clear fingernail polish to provide protection against moisture, and the vials are transferred to a - 7 0 ° freezer. The dimethyl sulfoxide promotes uniform freezing and prevents the formation of large ice crystals which could disrupt cell membranes. Strains are retrieved by scraping the surface of the frozen stock with a sterile stick and using the stick to inoculate a petri dish or culture tube. In this manner, the same stocks can be used repeatedly without thawing. We have found that stocks prepared in this manner maintain close to 100% viability for at least 15 years. It is important that cultures for frozen stocks be grown to stationary phase since frozen stocks made from log-phase cultures begin losing viability within the first year of storage. Mutant Isolation Many kinds ofC. c r e s c e n t u s mutants can be obtained without mutagen¢sis. Antibiotic-resistant mutants can be obtained by direct selection, and motility mutants can be obtained by serial enrichment procedures for nonmotile cells on soft afar plates. 7 Auxotrophic mutants can be obtained after enrichment for nongrowing cells. Since wild-type C a u l o b a c t e r is resistant to penicillin, however, fosfom~cin or D-cycloserine must be used instead. 4 The standard procedures described for penicillin enrichment are used for either of the two antibiotics, and prior mutagenesis is unnecessary. Cultures are grown in PYE, centrifuged, and resuspended in defined medium. After one doubling has occurred in defined medium, either 100 ~g/ ml D-cycloserine or 500/.~g/ml fosfomycin is added. Samples arc removed 13, 15, and 18 hr after the addition of antibiotic, spun, and resuspended to remove the antibiotic and plated on PYE medium. The resulting colonies are replicated onto defined medium to identify those with nutritional requirements. Auxotrophs are 5 to 10% of the survivors in typical experiG. Cohen-Bazire, W. R. Sistrom, and R. Y. Stanier, J. Cell. Comp. Physiol. 49, 25 (1957). 7 R. C. Johnson and B. Ely, J. Bacteriol. 137, 627 (1979).
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GENETICS
OF Caulobacter crescentus
375
ments. Auxotrophs and motility mutants can also be obtained by transposon mutagenesis (see below). Generalized Transduction
Generalized transduction involves the use of a bacteriophage to transfer genetic markers from one strain to another. For C. crescentus the bacteriophage tbCR30 is excellent for this purpose since it can transfer large pieces of DNA and is easy to g r o w ) The basic strategy is to grow tbCR30 on the donor strain to allow the random mispackaging of fragments of the donor DNA into bacteriophage particles. The resulting lysate is irradiated with ultraviolet light to inactivate the viral genomes and then stored over chloroform to kill any residual bacteria. Transduction experiments are performed by mixing a dilution of the irradiated donor lysate with the recipient strain on the surface of a petri dish containing a selective medium. Colonies which grow should contain the selected marker from the donor.
Preparation of Transducing Lysates An overnight culture of the strain chosen to be a donor is prepared, and 0.2 ml of this culture is mixed with 0.1 ml of a 10-4 dilution of ~bCR30 lysate (unirradiated) in 3 ml of melted PYE soft agar. The mixture is immediately poured onto the surface of a fresh PYE plate and allowed to harden. After overnight incubation at 33° , overlapping plaques should be observed. If the lysis appears satisfactory, 5 ml of PYE broth is pipetted onto the petri dish, which is refrigerated overnight to allow the bacteriophage to diffuse out of the agar into the liquid overlay. The next day, the broth is decanted into an empty 60-ram-diameter petri dish. The cover is removed and the contents are swirled under a germicidal ultraviolet light at a distance of approximately 45 cm for 5 min. (If the petri dish is swirled by hand, protective gloves should be worn.) After irradiation, the contents of the dish are poured into a glass screwcapped tube, a few drops of chloroform are added, and the tube is shaken vigorously to ensure that all parts of the tube come in contact with the chloroform. A drop of this lysate is placed on a PYE plate and incubated to verify that all of the contaminating bacteria have been destroyed. A good lysate should have a titer of more than 101°plaque-forming units (pfu) per milliliter before irradiation and less than 105 pfu/ml after irradiation. Although loss of transducing ability occurs slowly at 4 °, phage lysates can be refrigerated for several years and still maintain good donor ability. 8 B. Ely and R. C. Johnson, Genetics 87, 391 (1977).
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Transduction
Transduction experiments are performed by mixing 0.1 ml of a fresh overnight culture of the recipient strain with 0.1 ml of a 10 -2 or 10 -3 dilution of the donor lysate. The mixing is done on the surface of a petri dish containing the appropriate selective medium. Colonies appear after incubation for 2 to 5 days at 33 °. In the case where tetracycline resistance is used as a selective marker, a 10 ml overlay of melted PYE agar is poured on top o f a PYE plate containing I mg/ml tetracycline, and the plate is used as soon as the overlay hardens. The overlay allows time for expression of tetracycline resistance before sufficient tetracycline comes in contact with the recipient cells to cause cell death. When transduction experiments are performed on minimal medium, ~bCR30 growth is inhibited and phageresistant transductants are rarely observed. When transduction experiments are performed on a PYE medium, however, phage replication occurs, and a significant proportion of the transductant colonies will have mutated to phage resistance. In either case, 102 to 104 transductants are obtained per plate depending on the titer of the original donor phage lysate and the recipient marker being transduced. Conjugation Conjugation involves the transfer of DNA from one cell to another through direct cell contact. In the case of C. crescentus, this transfer can be mediated by the conjugative plasmid R P 4 . 9 RP4 is a broad host range plasmid which can be transferred among many gram-negative bacteria and will mediate intraspecies chromosomal transfer. Donor strains containing the RP4 plasmid produce pili which establish contact with the recipient strain and provide a conduit for the actual transfer of DNA from the donor to the recipient. Plasmid Transfer Plasmid transfer is an efficient process which can be accomplished by simply allowing contact between the donor and recipient strains. The simplest method is the cross-streak method. 1° A selective medium is chosen which will inhibit growth of both the donor and the recipient strains but will allow growth of those recipient cells which obtain the plasmid from the donor. Overnight cultures of both strains are prepared, and using a 0.1-ml pipette, approximately 20/.d of the donor culture is streaked 9 B. Ely, Genetics 91, 371 (1979). 1o B. Ely, Genetics 78, 593 (1974).
[17]
GENETICS OF C a u l o b a c t e r c r e s c e n t u s
377
across the surface of a petri dish containing a selective medium. The culture is drawn into the pipette by capillary action, and the pipette is held like a pencil to draw a line of culture fluid across the plate. After the fluid in the donor streak has soaked into the medium, streaks of one or more recipient strains are made in a similar manner, perpendicular to the donor streak. As the pipette containing the recipient culture crosses the donor streak, donor cells are dragged along and mixed with the recipient cells. After incubation for 2 days, growth is observed throughout the half of the recipient streak where mixing of the two strains occurred. The other half of the recipient streak and all but the intersection of the donor streak serve as controls to monitor for spontaneous mutation or contamination. Plasmid-containing recipient strains are purified by streaking for single colonies on selective medium. Chromosome Transfer
Chromosome transfer is less efficient than plasmid transfer and requires a longer time for the actual transfer process to occur. To maximize the efficiency of transfer, the donor and recipient strains are held together on a solid support while the transfer occurs. The filter mating procedure 9 is accomplished by mixing 0.1 ml of an overnight culture of the donor with 0.5 ml of an overnight culture of the recipient, then filtering the mixture through a sterile 2.5-cm-diameter 0.45-txm filter. After the excess liquid is removed, the filter is placed on a fresh PYE plate and incubated at 33° for 2 to 3 hr. The filter is then placed in a tube containing 1 ml of PYE broth and agitated vigorously to resuspend the cells. Aliquots (0.2 ml) of the resuspended cells are spread on the surface of petri dishes containing a medium which is selective for both the donor and the recipient cells. Generally 50 to 200 colonies appear after 3 to 5 days at 33 °. Transfer of up to one-sixth of the C. crescentus chromosome can be detected with this procedure.
Transposon Mutagenesis Transposition of both Tn5 and Tn7 occurs in C. crescentus. H However, Tn7 inserts at a single site on the chromosome and, therefore, is of limited usefulness for genetic studies.12 O n the other hand, Tn5 and its derivatives appear to transpose randomly and provide a means for obtaining a selectable marker in or near any C. crescentus gene. 11 B. Ely and R. H. Croft, J. Bacteriol. 149, 620 (1982). l: B. Ely, J. Bacteriol. 151, 1056 (1982).
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OTHERBACTERIALSYSTEMS
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Isolation o f New Transposon Insertions
Transposon mutagenesis occurs after the transposon is introduced into target strain on a vector which is unstable in C. crescentus. ~1 P-type plasmids containing bacteriophage M u 13 o r based on a ColE1 replicon TM provide excellent vehicles for the transient insertion of a transposon. Overnight cultures of the recipient strain and an E. coli strain containing the donor plasmid are prepared, and the filter mating procedure is carried out as described for conjugation experiments. Approximately 30 to 100 antibiotic-resistant colonies are obtained per plate, and each represents an independent transposition event. If a plasmid containing the Tn5 derivative Tn5-VB32 is used, 15promoter fusions can be obtained. Tn5-VB32 contains a promoterless neomycin phosphotransferase II (NPTase II) gene near one end which can be expressed from an adjacent promoter. 16If Tn5-VB32 is inserted into a C. crescentus gene in the proper orientation, the NPTase II gene is expressed from the promoter of that gene and serves as a reporter to monitor the expression of the mutated gene.17
Identification of Transposon Insertions Linked to Specific Markers
To obtain transposon insertions linked to a specific marker, the random mutagenesis procedure is used to generate approximately 500 colonies containing independent insertions. The resulting colonies are mixed together and suspended in PYE broth. An aliquot of the resulting suspension is then used as the host for the preparation of a ~bCR30 lysate as described above. Transduction experiments can be performed using the resulting lysate with selection for the antibiotic resistance encoded by the transposon. Subsequent screening for the loss of a recipient marker results in the identification of transposon insertions which are linked to the marker of interest. Phage lysates can then be prepared from individual transposon mutants, and transduction experiments can be performed to determine the proximity (linkage) of the transposon to the gene interest.
13j. E. Behringer, J. L. Brynon, A. V. Buchanon-Wallaston, and A. W. B. Johnston, Nature (London) 276, 663 (1978). 14 B. Ely, Mol. Gen. Genet. 200, 302 (1985). t5 V. Bellafatto, L. Shapiro, and D. A. Hodgson, Proc. Natl. Acad. Sci. U.S.A. 81, 1035 (1984). 16R. Champer, R. Bryan, S. L. Gomes, M. Purucker, and L. Shapiro, Cold Spring Harbor Syrup. Quant. Biol. 50, 831 (1985). 17 R. Champer, A. Dingwall, and L. Shapiro, J. Mol. Biol. 194, 71 (1987).
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GENETICS OF Caulobacter crescentus
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Exchange of Transposon-Encoded Antibiotics Resistance Sometimes it is useful to change the antibiotic resistance encoded by a specific transposon insertion. TM The kanamycin resistance encoded by Tn5 can be exchanged for tetracycline resistance using pBEE132 to introduce the Trd-132 into the Trt5-containing strain. Transfer of pBEE132 is accomplished by the filter mating procedure described above. Colonies which are tetracycline resistant and kanamycin sensitive have the tetracycline resistance of Tn5-132 substituted for the kanamycin resistance of the inserted Tn5 element. Electroporation Electroporation involves the use of a brief electric shock to depolarize the cell membrane and allow the uptake of DNA. Electroporation works well with C. crescentus for the uptake of plasmid DNA using procedures developed for E. coli. 19 However, in contrast to the case for E. coli, experiments with cut and religated plasmid DNA have not been successful. To prepare cells for electroporation, a 500-ml culture is grown at 33° to mid-log phase in PYE. The culture is divided into two centrifuge bottles and spun at 6000 g for 5 min. The pellets are resuspended in 100 ml distilled water, spun at 6000 g for 5 rain, and then resuspended in 100 ml of distilled water a second time and spun as before. The washed pellets are resuspended in l0 ml of 10% glycerol, combined and transferred to a 50ml centrifuge tube, spun at 3000 g for 5 rain, and resuspended in 2 ml of 10% glycerol. The concentrated cells are divided into 200-/.d aliquots and frozen at - 7 0 ° . For electroporation, a tube is thawed, and 40/zl of cells is placed in a microfuge tube along with 0.1 to 1/~g of DNA in a small volume of distilled water (or very dilute buffer). After 1 rain on ice, the mixture is transferred to an ice-cold Bio-Rad (Richmond, CA) Gene Pulser cuvette with a 0.2cm electrode gap and subjected to a 2.5-kV shock with the capacitor set at 25/~F (microfarad) and the resistance on the pulse controller set at 200 ohms. Under these conditions, the time constant is 5 msec. After this treatment, the contents of the cuvette are transferred to a culture tube containing 1 ml of PYE, and the cells are incubated at 33 ° with aeration for 1 hr. After the recovery period, the cells are plated on the appropriate selective medium. We routinely obtain 104 to 105 transformants per microgram of DNA. 18 B. Ely and T. Ely, Genetics 123, 649 (1989). 19 A. K. Ellgaard, D. A. Mullin, and S. A. Minnich, Abstr. Annu. Meeting Am. Soc. Microbiol., p. 208 (1989).
380
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Pulsed-Field Gel Electrophoresis Pulsed-field gel electrophoresis (PFGE) allows one to separate very large DNA fragments. A genomic map of the C. crescentus chromosome has been generated by aligning DraI, AseI, and SpeI restriction maps with the genetic map. ~s Therefore, the map location of transposon insertions can be determined by PFGE. This procedure is approximately 100 times faster than using a combination of conjugation and transduction to determine map locations. 2° A current version of the genomic map is shown in Fig. 1.
Preparation o f DNA Plugs DNA plugs are prepared by a modification of the method of Smith and Cantor. 2~A 2.5-ml aliquot of an overnight culture of a C. crescentus strain is spun at 4000 g in a refrigerated centrifuge, and the resulting pellet is resuspended in 2 ml of 10 mM Tris, pH 7.6, containing 1 M NaCI. The mixture is spun a second time, resuspended in 1 ml of the same buffer, and heated to 37 °. One-half of the suspension is then mixed with an equal volume of melted I% (w/v) agarose (PFG grade) previously equilibrated to 65 ° . The mixture is immediately transferred to plug molds and allow to cool. After cooling, each set of plugs is removed from the mold and transferred to a disposable plastic test tube (15 × 100 mm) which contains 1.8 ml of 1% (w/v) sarkosyl in 0.5 M E D T A (pH 9-9.4) and 0.2 ml of 10 nag/ ml proteinase K. After overnight incubation at 50°, the liquid is removed by aspiration and replaced with 2 ml of 10 mM Tris, pH 7.5, 1 mM EDTA and 30 tzl of a phenylmethylsulfonyl fluoride (PMSF) stock (17.4 mg/ml in 100% ethanol). After 1 hr on a rotator at room temperature, the liquid is removed by aspiration and replaced by an identical mixture. After a second hour, the procedure is repeated 3 times without the PMSF. After the final wash, the tube containing the plugs is refrigerated until needed. We have found that DNA-containing plugs can be stored in the refrigerator for several years.
Pulsed-Field Gel Electrophoresis Techniques PFGE experiments are carried out by digesting DNA-containing agarose plugs with restriction enzymes which cut the genome infrequently. The resulting DNA fragments are then resolved by insertion of the plug into an agarose gel and subjecting the DNA to PFGE. Restriction digests are carried out by placing a small segment of a DNA-containing agarose 20 A. Dingwall, L. Shapiro, and B. Ely, Methods 1, 160 (1990). 21 C. L. Smith and C. R. Cantor, Cold Spring Harbor Syrup. Quant. Biol. 51, 115 (1986).
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GENETICSOF Caulobacter crescentus
381
4000
trpE aroF I
l~c cysE
thi eux
metD 3500 hisB I
~10
500
-gO ' 0- ' ~ ~ c y s B
hisD
3000 aroG
t000 hunA pilA
aroC ,/ serA
3O5 hung trpB
,
gltA
~)/ I 15oo proA leuA
cysA hisG 2000
FIG. 1. Caulobacter c r e s c e n t u s genonlic map. Inner Circles show fragmentsgenerated by DraI, AseI, and SpeI restrictiondigests, respectively,with SpeI fragmentsrepresented on the innermostcircle. Fragment sizes are indicatedin kilobases. plug in a microfuge tube along with approximately 10 units of restriction enzyme, 10/.d of 10 x restriction buffer, and 50/zl distilled water. After incubation at 37° for 1 hr, the plug fragments are removed with a spatula and inserted directly into the gel wells for PFGE. Table I shows conditions which give good resolution of various size ranges of DNA fragments. After electrophoresis, the gel is stained with ethidium bromide and photographed. DNA fragments containing a Tn5 insertion can usually be identified by the altered migration of the corresponding band on the gel. Differences as small as 2% of the size of the fragment can be resolved under optimal conditions. Alternatively, the DNA can be blotted to nylon membranes and bands identified by hybridization.
382
orrtEa BACTERIALSYSTEMS
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TABLE I CONDITIONS FOR SEPARATING VARIOUS SIZE RANGES OF DNA FRAGMENTS BY PULSED-FIELD GEL ELECTROPHORESIS Fragment size range (kb)
Pulse time (sec)
Voltage (V)
Time (hr)
5-50 20-100 50-250 100-400 200-1000
1 3 10 20 45
200 200 200 200 200
10 10 14 14 16
Complementation Complementation experiments involve the transfer of cloned DNA into a mutant strain to determine if the transferred DNA can correct the mutant phenotype. Positive results can be obtained as a result of complementation, recombination, or reversion. False positives arising from reversion are detected by the use of appropriate controls, and recombination can be prevented by the use of a recipient strain which is rec-.22 Construction o f rec- Strains Strains defective in recombination are constructed by use of strain PC7070 for cotransduction of the recA526 allele with a linked Tn5 insertion. 23Phage grown on PC7070 is used to transduce a kanamycin-sensitive strain containing the mutation to be complemented. The presence of the recA526 allele is demonstrated by an increased sensitivity to irradiation with ultraviolet light. Strains containing the mutation are sensitive to a 30sec exposure to a germicidal lamp at a distance of 45 cm, whereas those containing the wild-type allele are resistant to this exposure. In practice, a master plate of transductant colonies is replicated onto two plates. One of the replicas is exposed to the ultraviolet light, and both are incubated overnight. Colonies which grow only on the unirradiated plate contain the rec allele. Alternatively, the recA allele can be transferred by cotransduction with the cysB gene (2% linkage) in order to avoid the use of a Tn5 insertion (A. Newton, personal communication, 1978). 22 E. A. O'Neill, R. H. Hynes, and R. A. Bender, Mol. Gen. Genet. 198, 275 (1985). 23 N. Ohta, M. Masurekar, D. A. Mullin, and A. Newton, J. Bacteriol. 172, 7027 (1990).
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GENETICS OF Caulobacter crescentus
383
Complementation o f rec- Strains
Complementation experiments are performed using the cross-streak method described earlier. Complementation of a nutritional mutation can be observed directly by performing the transfer on defined medium. Complementation of motility mutations or other mutations where direct selection is not possible is done by using antibiotic resistance to select for transfer of the plasmid containing the cloned DNA. The plasmid-containing strain is then purified and checked for the loss of the mutant phenotype. Positive complementation results can be verified by growing the strain under nonselective conditions and screening for loss of the plasmid. The resulting strains are then tested for the reappearance of the mutant phenotype. Vectors based on RSF1010 are lost very quickly in C. crescentus, so few colonies will contain the plasmid after plating an aliquot of a culture grown overnight on PYE broth. In contrast, when the same procedure is used with vectors based on pRK290, approximately 1% of the resulting colonies have lost the plasmid, so a greater number of colonies need to be screened to obtain the desired segregants. Inactivation of Chromosomal Genes Chromosomal genes can be inactivated, or specific mutations can be introduced into the chromosome, through the use of cloned genes. The desired mutation is introduced into a cloned copy of the gene, and the plasmid containing the mutated gene is transferred to the appropriate C. creseentus strain. Recombination between the chromosomal and the plasmid copies of the gene usually results in the integration of the plasmid into the chromosome. Thus, the resulting strain will have both the wildtype and the mutated forms of the gene. Subsequent loss of the plasmid antibiotic resistance owing to a second recombination event results in the loss of one copy of the gene. Depending on where recombination occurs these segregants will have either the wild-type or the mutated version of the gene. Vectors for gene replacement should be incapable of replication in C. crescentus. Two types of vectors are appropriate. The first are vectors which contain the sequences necessary for mobilization by the IncP transfer system. 24These plasmids replicate well in E. coil and can be transferred from E. coil to C. crescentus at a high frequency 25(also B. Ely, unpublished observations, 1987). However, they cannot be replicated in C. crescentus, so they are lost unless recombination occurs to form a cointegrate. Trans24 R. Simon, U. Pfiefer, and A. Puhler, Bio Technology 1, 784 (1983). S. A. Minnich, N. Ohta, N. Taylor, and A. Newton, J. Bacteriol. 170, 3953 (1988).
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duction can then be used to obtain true gene replacements where the resulting strain contains a single copy of the mutated gene. 25A transducing lysate is prepared on a pool of cointegrate colonies and used to transduce the appropriate C. crescentus strain with selection for a marker either within the gene of interest or closely linked to it. The resulting colonies are then screened for the presence of the mutated gene and the absence of the plasmid antibiotic resistance. This procedure can also be used to transfer genes to a new chromosomal location using homology between Tn5 insertions for recombination (N. Ohta, L. S. Chen, D. A. Mullen, and A. Newton, personal communication). In this case, a plasmid containing a Tn5 element will integrate at the site of any chromosomal Tn5 insertion. A second procedure for the isolation of gene replacements is the use of electroporation to introduce the plasmid containing the mutated gene into the appropriate C. crescentus strain (J. Malakooti and B. Ely, unpublished observations, 1990). In this case, any vector can be used as long as it does not replicate in C. crescentus. After the plasmid containing the mutated gene is constructed in E. coli, plasmid DNA is isolated and used to transform the C. crescentus strain with selection for the plasmid antibiotic resistance. If ampicillin is used as the selective marker, a derivative of the ampicillin-sensitive strain SCl107 must be used as the host. The resulting colonies will be cointegrates, and gene replacements can be isolated as described above. Cointegrates are obtained at a frequency of 103 per microgram of plasmid DNA. This procedure also can be used to introduce a new restriction site into the genome at a particular location. Homologous recombination between a cloned fragment in the appropriate vector and the chromosome would result in the integration of the plasmid. If the plasmid contains one of the restriction sites used for PFGE mapping, the position of the cloned DNA could be readily determined as described above.
[18] G e n e t i c A n a l y s i s o f Agrobacteriurn
By GERARD A. CANGELOSI, ELAINE A. BEST, GLADYS MARTINETTI, and EUGENE W. NESTER
Introduction Genetic infection by Agrobacterium species is the only verified example of natural genetic exchange between the prokaryotic and eukaryotic kingdoms. Infection of plants by Agrobacterium strains containing tumorMETHODS IN ENZYMOLOGY,VOL. 204
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any formreserved.
