[13]
GENETIC ANALYSISIN Bacillus subtilis
305
[13] G e n e t i c A n a l y s i s in Bacillus subtilis
By JAMES A. HOCH Introduction Of the bacteria Bacillus subtilis is the easiest organism to manipulate genetically. The armamentarium of genetic tools includes PBS 1 transduction which transfers very large [> 150-200 kilobase (kb)] fragments of the chromosome and DNA-mediated transformation which utilizes smaller DNA fragments (40 kb or less). The chromosomal location of new markers can be rapidly determined by transducing a set of auxotrophic strains covering all regions of the chromosome.l This transduction system takes the place of conjugation, which has never been developed for this organism. DNA-mediated transformation occurs at high frequency and is most useful for fine structure analysis of chromosomal regions up to 10 kb. In addition, a protoplast transformation system has been developed to allow easy transfer of plasmids between strains. Strategic Considerations In a common scenario, an investigator has isolated a series of mutants known to affect a particular pathway or phenomenon and wishes to determine the number of loci involved and their chromosomal locations. The first step in determining the chromosomal locations of the loci is to transduce a set of strains with auxotrophic markers conveniently located around the chromosome such that the entire chromosome will be covered.l Figure 1 shows the genetic markers that may be conveniently used for such an analysis. A PBS1 transducing lysate is prepared on the strain of interest and used to transduce each recipient to test for linkage. In practice all of the crosses can be carried out in a single day and a general chromosomal location determined in a week. Once the general location of the locus is obtained, further fine structure analysis of its location should be carried out by transformation. A large variety of genetic markers exist in virtually all regions of the chromosome. 2 Before embarking on genetic studies in B. subtilis, it is strongly suggested that the classic paper by C. Anagnostopoulos and co-workers 3 be 1 M. A. D e d o n d e r , J. A. L e p e s a n t , J. L e p e s a n t - K e j z l a r o v a , A. BiUault, M. Steinmetz, and F. K u n s t , Appl. Environ. Microbiol. 33, 989 (1977). 2 p. j. Piggot a n d J. A. H o c h , Microbiol. Rev. 49, 158 (1985). 3 M. Barat, C. A n a g n o s t o p o u l o s , a n d A . - M . Schneider, J. Bacteriol. 90, 357 (1965).
METHODS IN ENZYMOLOGY,VOL. 204
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
306
OTHER BACTERIAL SYSTEMS thiC
[13]
purAguaAcysA ~ a!ol
ctr~~''r 360/0~ " ~ 1
hisA~o,.
- "
~purB
thrB-~ '~Tn
Off ~..glyB
,roo . . . . . aroD ~
pyrA
trPCm'-tB ~ t K~, gltA glnA sw/~ FzG. 1. Landmark loci of the B. subtilis chromosome. The chromosome is divided by
degrees. Strains bearingthese markers are used to locateunknownmarkersby transduction. Strains are availablefromthe BacillusGeneticStock Center, Ohio State University,Columbus, Ohio.
read. This publication is a virtual treasure trove of genetic information and techniques and explores in detail techniques such as the recombination index that are not explained here.
Transformation and TransductionProcedures
Solutions PAB: 17.5 g antibiotic medium 3 (Difco, Detroit, MI) dissolved in 1 liter deionized water. Autoclave. TBAB: 17.5 g tryptose blood agar base (Difco) and 8.5 g Bacto Agate (Difco) suspended in 1 liter deionized water. Autoclave and dispense 25 ml in petri dishes. MG: 2 g ammonium sulfate [(NH4)2SO4]; 14 g potassium phosphate, dibasic (K2HPO4); 6 g potassium phosphate, monobasic (KH2PO4); 1 g sodium citrate (Na3C6HsO7 • 2H20); 0.2 g magnesium sulfate (MgSO4 • 7H20). Dissolve each of the ingredients successively in 1 liter of deionized water. Autoclave. Add 10 ml of sterile 50% glucose. For MG plates make a 2 × solution of salts and a 2 × solution of agar (Bacto-agar, Difco) to give 1.5% final agar concentration. Autoclave separately. Mix at 50° and add 10 ml of 50% glucose per liter. Fifty percent of glucose is easily made by dissolving 500 g glucose in 750 ml distilled water.
GENETIC ANALYSISIN Bacillus subtilis
[13]
307
CH: 5.0 g casein hydrolyzate, acid (Difco), dissolved in 100 ml deionized water. Autoclave. Note that CH does not contain tryptophan, glutamine, purines, pyrimidines, or vitamins. Amino acid stock solutions: 5 mg/ml in deionized water. Autoclave.
First Growth Period Supplemented MG. The final concentration of additives is 0.02% CH and 50 t~g/ml of any amino acid purine or pyrimidine required by the recipient. For one transformation: 1. Dilute 0.1 ml of CH in 2.4 ml of MG. Add 0.25 ml to a 20 × 150 mm culture tube. 2. Dilute 0.1 ml amino acid stock solution in 0.9 ml of MG. Add 0.25 ml of each amino acid required by the recipient to the above tube. Add sufficient MG to bring the volume to 2.5 ml.
Second Growth Period Supplemented MG. The final concentration of additives is 0.01% CH and 5/xg/ml of required amino acid. I. Make a 1/10 dilution of the required amino acid dilution and a I/2 dilution of CH dilution from First Growth ,Period Supplemented MG. 2. To a 20 x 150 mm culture tube add 0.1 ml of CH, 0.1 ml of each amino acid dilution, 0.1 ml 50 mM MgSO4, and MG to 0.9 ml.
Chromosomal DNA Preparation Inoculate the donor strain lightly in 20 ml of MG supplemented with 20 t~g/ml amino acid requirements and 0.05% CH in a 250-ml culture flask. Grow overnight with shaking at 37°. Centrifuge the culture I0 min at 4 ° at 7000 rpm (SS-34 head in a SorvaU centrifuge). Resuspend the pellet in 2 ml of TE (10 mM Tris, 1 mM Na2EDTA , pH 8.0) in a 16 × 125 mm culture tube and add 5 mg/ml fresh lysozyme and 8/xl of a solution of RNase at 10 mg/ml. Incubate for 30 min at 37°. Add proteinase K to 1 mg/ml and incubate for 15 min at 37 °. Next add 200/xl of 10% sodium dodecyl sulfate followed by 2 ml of phenol saturated with TE. Shake gently for 1 min. Centrifuge to separate the layers and extract the top layer with 2 ml phenol-chloroform (1 : 1) followed by 2 ml chloroform. Four milliliters of 95% (v/v) ethanol is layered on the final supernatant, and the D N A is spooled out on a fine glass rod or Pasteur pipette. Dissolve the DNA in 200/zl of TE.
Transformation Transformation requires the preparation of a cell culture competent to take up DNA. This mystical state occurs at the beginning of stationary phase and is associated with the onset of sporulation. Some mutants are difficult to bring to competence and therefore transform poorly. Many
308
OTHER BACTERIAL SYSTEMS
[13]
times this is a result of poor growth in the First Growth Period, and the simple addition of a little yeast extract (e.g., 0.01%) will solve the problem, Presented below is the original recipe for transformation devised by C. Anagnostopoulos and Spizizen4; which works well on a wide variety of strains.
First Growth Period 1. Streak the recipient strain on a TBAB plate the evening before the experiment. Incubate at 37 °. 2. From the fresh overnight TBAB culture sufficient cells are taken to give a lightly turbid suspension in 2.5 ml of First Growth Period Supplemented MG in a 20 x 150 mm sterile culture tube. 3. Incubate with shaking at 37° for 4 hr. The culture should be quite turbid; if not, incubate an additional 1 hr.
Second Growth Period 1. Prepare two culture tubes for the second period by diluting 0. I ml of culture in 0.9 ml Second Period growth medium in each of two 20 x 150 mm tubes. 2. To one tube add 0.1 ml of DNA solution (1 to 0.0001 /zg DNA) diluted in MG. To the second tube (the reversion control) only add MG. 3. Incubate with shaking at 37° for 90 min. 4. Prepare serial dilution tubes containing 0.9 ml MG and dilute the transformation tube 1/I0, 1/100, and 1/1000. Spread on separate MG plates 0.1 ml of the undiluted culture or 0.1 ml of each dilution along with 0.1 ml of appropriate amino acid stock solution required by the recipient but not selected for in this experiment. 5. Spread 0. I ml of undiluted reversion control culture on appropriately supplemented plates. 6. Incubate plates at 37° for 48 hr. This standard transformation procedure will yield around 100 colonies on the 1/I000 dilution plate for highly competent cells at DNA saturation. This corresponds to a frequency of 1% transformation. DNA saturation under these conditions is around 0.I-1.0 mg DNA/ml.
Protoplast Transformation The introduction of replicating plasmids into B. subtilis is most efficient with plasmid multimers. Furthermore, if the plasmid contains an insert of DNA homologous to the B. subtilis chromosome, it is likely to be subjected 4 C. Anagnostopoulos and J. Spizizen, J. Bacteriol. 81, 741 (1961).
[13]
GENETIC ANALYSISIN Bacillus subtilis
309
to a recombination event that can cause loss of the insert or gene conversion. Therefore, under these conditions it is best to introduce plasmids into recipients using the protoplast transformation procedure. Presented below is a simplified version (M. Zukowski, personal communication, 1990) of the original Chang and Cohen procedure: The procedure works well with minipreparation plasmid DNA. Solutions 2 × SMM buffer: 1.0 M sucrose, 40 mM sodium maleate, 40 mM MgCI 2 . Adjust to pH 6.5 and autoclave. SMMP medium: Mix together equal volumes of 4 x PAB (autoclaved) and 2 x SMM (autoclaved). PEG solution: 40 g polyethylene glycol (PEG) 6000, 50 ml of 2 x SMM buffer. Bring the volume to 100 ml with deionized water and autoclave. DM3 Regeneration Medium 1. Mix, in 700 ml deionized water, 135 g sodium succinate, 5 g casamino acids (Difco), 5 g yeast extract, 1.5 g KH~POa, and 3.5 g K2HPO4. 2. Adjust to pH 7.2. 3. Bring the volume to 950 ml with deionized water. 4. Add 8 g agar. 5. Autoclave. 6. Cool to approximately 55 °. 7. Add 25 ml of 20% sterile glucose (preheated to 55°). 8. Add 20 ml of 1 M MgCI2 (preheated to 55°). 9. Add 5 ml of 2% bovine serum albumin (BSA, freshly prepared, filter-sterilized). 10. Add antibiotics as required: chloramphenicol, 10 tzg/ml final concentration; kanamycin, 100/xg/ml final concentration. 11. Pour plates, leave on the benchtop overnight, then store at 4 ° for no longer than 1 month. Procedure 1. A starter culture is grown overnight in 10 ml of PAB at 37° with shaking. 2. The following morning, 0.5 ml of starter culture is inoculated into 50 ml of PAB. Shake the culture at 37° until the cell mass reaches an OD660 of 0.4-0.5. 3. Transfer the culture into a 50-ml plastic centrifuge tube. Pellet the s S. Chang and S. N. Cohen, Mol. Gen. Genet. 168, 111 (1979).
310
OTHER BACTERIAL SYSTEMS
[13]
cells by centrifugation (e.g., I0 min at 6000 rpm in Beckman TJ-6 centrifuge with a fixed-angle rotor). 4. Resuspend the pellet in 5 ml of SMMP with 2 mg/ml lysozyme freshly added. The solution may be filter-sterilized before adding to cells. 5. Incubate the cells in a plastic centrifuge tube at 37° with gentle rocking (about 100 rpm) for 90 min (60 rain may be satisfactory in some cases). Microscopic evaluation of protoplasting efficiency may be called for, but loss of schlieren in the suspension should be indicative. 6. Pellet protoplasts by centrifugation (e.g., 10 min at 5000 rpm at 4° in Beckman TJ-6 centrifuge with a fixed-angle rotor). 7. Pour off the supernatant and add 2.5 ml of SMMP. Resuspend the protoplasts by vigorous agitation of the tube with fingers. Do not use a vortex.
8. a. Mix the plasmid DNA with an equal volume of 2 × SMM in a 50-ml plastic centrifuge tube. b. Add 0.5 ml of the protoplast suspension. Agitate the tube lightly with fingers. c. Add 1.5 ml of the PEG solution. Gently mix by rolling the tube manually. d. After 2 rain of exposure to the PEG, add 5 ml SMMP. 9. Repeat Step 6. I0. Resuspend the pellet in I ml of SMMP; incubate at 37° with gentle shaking for 90 min. 11. Plate 0.1-ml samples onto fresh DM3 regeneration medium plates to which antibiotics have been added as required. Incubate plates at 37° for 48-72 hr. Expect over 105 transformants//Lg for uncut DNA and 104-10 5 transformants//.~g for cut and ligated DNA. Expect no transformants if MgC12 was omitted from solutions.
PBS1 Transduction PBS I is a large transducing phage with the capability of encapsulating 150-200 kb of chromosomal DNA in the transducing particle. It depends on motile flagella for attachment and therefore is not useful for Fla-, Mot-, or any other strain with mutations that impair motility. The vast majority of researchers that use it have never seen a PBS1 plaque since it is an exceedingly poor plaque former. Despite these problems this transducing system works well for locating markers on the chromosome. PBS1 Stock Lysate. PBS1 stock lysates are prepared on the highly motile Bacillus licheniformis 8480 strain or any highly motile B. subtilis strain. Bacillus licheniformis 8480 (strain 5A1 in the Bacillus Genetic Stock Center Collection) cells from an overnight TBAB plate are inoculated at light turbidity into 2.5 ml of PAB in a 20 x 150 mm culture tube. Shake
[13]
GENETIC ANALYSISIN Bacillus subtilis
311
the tube at 37° for 2.5 hr. Add 0.1 ml of PBS1 stock lysate; allow adsorption for 5 min at room temperature. Dilute the tube into 25 ml of PAB in a 250ml culture flask. Shake at 37° for 2 hr. Place the flask in 37° incubator overnight without shaking. Centrifuge the lysate at 10,000 g for 20 min at 4° to pellet debris. Add 50/zg/ml DNase I (dissolved in 0.01 M MgSOa) and incubate 30 min at room temperature. Filter-sterilize the supernatant through a 0.45-/~m filter. Store at 4°. The lysate will be stable for at least 1 year. Preparation of Donor Lysates. The donor strain is streaked on a TBAB plate and grown overnight at 37°. Donor cells are inoculated at light turbidity (OD540 -0.1) into 2.5 ml of PAB in a 20 × 150 mm culture tube. Shake at 37° for 2.5 hr. The cells at this time are just beginning to become motile. Add 0.1 ml of the PBS 1 stock lysate. Incubate at room temperature for 5 min. Dilute into 25 ml of PAB in a 250-ml culture flask. Incubate with shaking at 37° for 1 hr. Add chloramphenicol to 5/~g/ml (spectinomycin or no antibiotic at all may be used if the strain is CmR). Shake for an additional 2 hr at 37°. Let the culture stand without shaking at 37° overnight. Centrifuge the lysate at 10,000 g for 20 min at 4° to remove debris. Add 50/~g/ml DNase I and incubate at room temperature for 30 min. Filtersterilize through a 0.45/.tm filter. Store the lysate at 4°. Although this procedure seems empirical, and is, it works almost every time, and after several times it seems natural. It is important to treat the lysate with DNase I because it will not filter well without it, and this step avoids transformation of the recipients. Transduction Procedure. The recipient strain is grown overnight on a TBAB plate at 37° and inoculated at light turbidity into 2.5 ml of PAB in a 20 x 120 mm culture tube. Shake at 37° for 4 hr. The cells at this stage should be motile and uniform in size when examined microscopically. Add 0.5 ml of cells and 0.5 ml of donor lysate to a 16 × 125 mm culture tube. To a second tube add 0.5 ml cells and 0.5 ml of PAB for a reversion control. Shake both tubes for 20 min at 37°. Plate 0. l ml of transduction and reversion cultures on appropriately supplemented MG plates for selection of recipient markers. This procedure normally gives between 50 and 500 transductants on the selection plate. Since 0.1 ml of rich medium is plated on the MG plates a slight lawn appears on the plate. This can be a problem with certain markers such as vitamin deficiencies, and, if so, the transduced cells can be centrifuged and washed with MG before plating. Analysis of Genetic Data The basic measure relating genetic and physical distance between two markers in transduction and transformation is the recombination unit. This is defined as percent recombination = 100 (1 - cotransfer), where
312
OTHER BACTERIAL SYSTEMS
[13]
degrees
1.0
5.5
\
I1.0
16.6
0:4
016
22.1
27.6
i
0.8 0.6 C
\,
\
x\
0.4
x~
0.2 X""--
012
/
X...._ X ~
0.8
| -
I.(3
FIG. 2. Relationship between cotransfer values in transduction and physical distance between markers on the chromosome. A graph of the Kemper formula C = (1 - t) + t(ln t) relates cotransfer values to the degrees separating two markers on the chromosome. 5,6 C is the cotransfer value between markers, and t is the length of the PBSI transducing DNA fragment.
cotransfer of linked markers is the number of double transformants divided by the single transformants. Markers further apart on the chromosome will have a smaller cotransfer value and a larger recombination value than closely linked markers. To be meaningful in terms of the physical distance separating two markers, the recombination values can be converted to physical distance using the Kemper formula, C = (1 - t) + t(ln t), where C is the cotransfer value and t is the fractional length of the donor DNA separating the two markers. 6 If the average size of the donor DNA is known (T), the physical distance between the two markers (D) can be calculated from D = tT. F i g u r e 2 7 shows the Kemper formula in graphical form where the t value in PBS I transduction has been related (approximately) to the degrees separating the markers on the chromosome. The graph makes it clear that cotransfer or recombination values are not additive. For example, a marker, X, located equidistant between two outside markers, A and B, and showing 50% recombination to each would be about 0.2t or 5.5 ° from each. The recombination values expected between A and B markers would not be 100% (50% + 50%) but rather 75% (0.2t + 0.20. The assumptions and calculations behind this figure have been detailed elsewhere. 7 The Kemper analysis is most useful in placing markers on the chromosome 6 j. Kemper, J. Bacteriol. 117, 94 (1974). 7 D. J. Henner and J. A. Hoch, Microbiol. Rev. 44, 57 (1980).
[13]
GENETIC ANALYSISIN Bacillus subtilis
313
5.00
i
15
4.25
3.50
u)
2.75
0 .J
2.00 -4
I
I
I
I
-3
-2
-1
0
Log ~
0 1
{]t.Q/ml)
FIG. 3. Relationshipbetween singleand double transformationand DNA concentration in transformationof weaklylinked markers. The data (fromRef. 2) showthe Leu÷ (©) and Phe÷ Leu÷ (A) transformantsfor the weaklylinked markersphe-1 and leu-1.
map by PBS1 transduction where the size of the transducing fragment is thought to be uniform. In transformation, the size of the transforming DNA can vary greatly from preparation to preparation, and the values for C are size dependent, particularly the lower values. In general 7-10% recombination between two markers in transformation means that about 1 kb of DNA separates the two markers. The interested reader is referred to Ref. 3 for detailed examples of linkage analysis using both systems and to Henner and Hoch 6 for an in-depth study of the application of the Kemper formula, Congression and Strain Construction Congression is a phenomenon unique to transformation and refers to the uptake of more than one transforming DNA fragment by a competent cell, particularly at high DNA concentrations. Congression is a two-edged sword in that it disturbs linkage analysis of distant markers but is extremely helpful in strain construction. Figure 3 shows the effect of congression on the weakly linked phe and leu markersJ As the DNA concentration reaches saturating levels for the competent cell population, the number of double transformants Phe + Leu + increases greatly in the cross wild-type
314
OTHER BACTERIAL SYSTEMS
[13]
DNA x phe leu recipient. The recombination values between closely linked markers are not subject to this effect. The example shown in Fig. 3 is of weakly linked markers where the recombination value stabilizes at nonsaturating DNA concentrations. Completely unlinked markers show no cotransfer at nonsaturating DNA concentrations. Congression is the major technique used for strain construction if unlinked markers are involved. Any selectable marker in the recipient can be exchanged for one in the donor by transformation using DNA concentrations greater than 1/xg/ml. For example, a trpC2 leu-1 recipient could be changed to a phe-1 leu-1 strain by transforming with DNA containing the phe-I allele with selection for Trp ÷ and searching among the Trp + transformants for Phe- recombinants. Piggot and de Lencastre have described a method to generate multiple marked strains where the recipient does not lose the selected marker. 8 This method is based on the observation that the products of certain early sporulation genes, for instance, spolIA, are required only in the mother cell and not in the developing forespore. Thus, if one transforms a spolIA recipient for Spo ÷ with wild-type DNA and selects for Spo ÷, some of the transformants will be genotypically spolIA because the transforming Spo ÷ DNA integrated into the chromosome that remained in the mother cell and, therefore, allowed sporulation to occur but did not convert the forespore chromosome to Spo ÷. In practice, one can transform a spolIA recipient with saturating phe-1 DNA, for example, selecting Spo + and searching for phe-1 spolIA recombinants among the transformants. Successive markers can then be added to this strain by repeating the procedure with new donor DNA. Thus, congression is a powerful tool to facilitate strain construction with characterized markers. It is also a simple and important means to backcross primary mutant strains to determine if all the phenotypes of a mutant can be ascribed to a single mutation.
Studies of Complementation and Dominance Using Integrative Vectors Since both complementation and dominance may reflect the subtle interplay of regulatory elements, the vectors to study these phenomena were designed to balance the concentration of the elements involved. The majority of these studies utilize integrative vectors which are basically Escherichia coli plasmids carrying an antibiotic resistance gene that is expressed in B. subtilis. One of the simplest and most utilized of these vectors is pJH101, which has a CAT (chloramphenicol acetyltransferase) g P. J. Piggot and H. de Lencastre, J. Gen. Microbiol. 106, 191 (1978).
[13]
GENETICANALYSISIN Bacillus subtilis
315
NdeI-Sau 3A)
Xm°,,,f..;-
~,~~,,~j~,(NdeI-Hpa
II)
Hind III c/orang site
FIG. 4. Map of the integrative vector pJMl03. The vector was constructed by ligating a filled-in H p a l I - S a u 3 A fragment of pC194 IS. Horinouchi and B. Weisblum, J. Bacteriol. 150, 815 (1982)] into the NdeI site ofpUC19. A sister plasmid (pJMl02) was also constructed from pUCI8 (M. Perego and J. A. Hoch, unpublished, 1988).
gene in the PoulI site of pBR322. 9 Integrative vectors are incapable of replication in B. subtilis and therefore do not transform B. subtilis to chloramphenicol resistance. If a region of homology to the chromosome is cloned on the vector, transformation results in chloramphenicol-resistant colonies which are so-called Campbell recombinants where the inserted vector is flanked by a duplication of the original chromosome fragment carried on the vector. As long as this fragment is big enough to prevent interruption of the transcription unit being studied, meaningful complementation and dominance results between alleles can be obtained since there are only two copies of the region being studied on the chromosome. The vector is also quite stable when integrated. Figure 4 shows a newer integrative vector, pJM103, which has a CAT gene in pUC19. Kanamycin-resistant derivatives of pUC18 and pUC19 are also available (M. Perego and J. A. Hoch, unpublished, 1988). pJM103 has all the properties of pUC19 in E. coli, therefore cloning of B. subtilis fragments may be carded out in E. coli utilizing the blue/white Lac selection system. After cloning and characterization of the DNA fragment of interest, the plasmid preparation is used to transform B. subtilis using the competence regimen outlined above. Minipreparations of the plasmid work very well. 1° Chloramphenicol resistance (CmR) may be selected on 9 F. A. Ferrari, A. Nguyen, D. Lang, and J. A. Hoch, J. Bacteriol. 154, 1513 (1983). i0 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979).
316
OTHER BACTERIAL SYSTEMS
[13]
any enriched medium such as TBAB containing 5/xg/ml chloramphenicol. Cm R transformants are readily obtained with DNA inserts as small as 400-500 base pairs (bp), but the yield falls rapidly below this size range. The smallest DNA insert that we have found to give a Campbell recombinant is 149 bp. Integrated vectors may be treated as a locus in transformation and transduction studies. PBS 1 transduction may be used to locate the vector on the chromosome. In transformation studies the vector plus duplication is a significant fraction of the transforming DNA segment, and therefore larger DNA fragments (i.e., gently prepared DNA) are required to show linkage to closely linked markers. Recombination values are depressed because of the longer DNA fragments, and severe recombinational polarity is observed. Polarity results from the fact that any shearing within the vector in the DNA preparation results in loss of the Cm R recombinant. Because of these complications, recombination values in transformation are not a reliable indicator of the location of integrated plasmids. A plasmid system for complementation and dominance studies has been constructed to allow integration in the SP/3 prophage found in all B. subtilis strains. II This plasmid, pFH7, contains a region of homology to SP/3 and integrates into it on transformation in B. subtilis. The advantages of this system are that the gene of interest is inserted in another location in the chromosome, without disturbing its normal location, and induction of the prophage results in a specialized transducing phage carrying the gene. Mutagenesis and Cloning with Integrative Vectors Integrative vectors are highly mutagenic if the insert chromosomal DNA fragments are wholly contained within a gene or transcription unit. Both copies of the gene or transcription unit generated by the integration event are disrupted in this case. Insert fragments that stop within a gene or transcription unit and extend beyond either of its ends are nonmutagenic since one good copy of the gene is generated in the integration event. In a random mutagenesis experiment using TaqI chromosomal fragments cloned in the AccI site of pJM102, 1% auxotrophs and 10% sporulation mutants were obtained among the Cm R transformants (M. Perego, unpublished data, 1989). Mutations generated in this fashion allow the affected locus to be easily cloned by excision of the vector and adjacent sequences with restriction enzymes, ligation in vitro, and transformation in E. coli. The procedure for recovery of integrated plasmids is straightforward. 11 E. Ferrari and J. A. Hoch, Mol. Gen. Genet. 189, 321 (1983),
[13]
GENETIC ANALYSIS IN Bacillus subtilis
Hindlll
I
EcoRI
H
Hlndlll
317
EcoRI
I I
E
I
FIG. 5. Integration of pJM103 in the chromosome. The hypothetical genes labeled A - F are adjacent to the cloned fragment X-Y. The fragments H and E are the expected plasmids recovered by HindlII and EcoRI digestions, respectively.
An overnight culture of the strain containing the integrated vector is used to prepare chromosomal DNA as described above. Two micrograms of this DNA is digested with the appropriate restriction endonuclease in 30 /.d of the appropriate buffer for the enzyme used, and 1/xg is removed to check the digestion on an agarose gel. The remaining 15 t~l is diluted to 100 /zl with deionized water and extracted with phenol followed by a chloroform extraction. The aqueous layer (-80/zl) is diluted with ligation buffer to 100/zl, ligase (1 unit) is added, and the mixture is incubated at 15° overnight. The entire ligation mixture is used to transform E. coli DH5a with selection for ampiciUin resistance. Usually 20-50 transformants are obtained. Figure 5 shows an example of the insertion of pJM 103 into a cluster of genes and the expected plasmids that would be obtained by excision with EcoRI (E) or HindlII (H). Any restriction enzyme with a site within the multilinker ofpJM103 can be used, but the direction of walking will depend on the orientation of the site to the inserted fragment. Gene disruption experiments using integrative vectors are standard procedure for the analysis of cloned DNA fragments where the phenotype of a disrupted open reading frame of unknown function is in question. Mutagenesis by integrative vectors compares favorably in frequency with the best classic mutagenesis procedures. It can be used in a random procedure using total chromosomal DNA or directed specifically to a
318
OTHER BACTERIAL SYSTEMS
[13]
cloned region or gene. The locus of the mutagenic plasmid can be readily cloned from the affected strain. Mutation with Chemical Agents
Bacillus subtilis is susceptible to all the chemical mutagenic agents used for induction of mutations in other bacteria. However, a very effective method of mutagenesis unique to spore formers was described by Balassa.12 If spores are germinated in the presence of chloramphenicol to prevent protein synthesis, they are extremely susceptible to N-methyl-N'nitro-N-nitrosoguanidine mutagenesis. Up to 25% sporulation mutants and 5% auxotrophs were found among the survivors of such treatment. This procedure is probably applicable to any sporulating bacterium and to a variety of mutagenic agents. Procedure. A suspension of spores of the strain of interest is prepared in a sporulation medium such as AK agar (BBL, Cockeysville, MD) by streaking the strain on the agar plate, incubating at 37° for 48 hr, and harvesting the spores by scraping the plate with 5 ml of PAB. Vegetative cells are killed by the addition of a drop of chloroform, and the suspension is stored at 4 °. Fifty milliliters of PAB containing I00/~g/ml chloramphenicol in a 250 to 500-ml culture flask is inoculated with the spore suspension to give 107- l0 s spores/ml. The suspension is incubated with shaking at 37° for 1 hr to give 90-99% germination. Germinated spores turn phase-dark in a phase microscope in contrast to the highly refractile ungerminated spores. N-Methyl-N'-nitro-N-nitrosoguanidine is added to a final concentration of 50/zg/rnl, and incubation is continued for 30 rain. The suspension is then centrifuged 6000 g for 15 rain at 4 ° to pellet the spores, and the treated spores are resuspended in sterile MG and stored at 4 °. Dilutions of the treated spores are plated on the desired medium for mutant selection or screening. This procedure is extremely effective when used correctly. The amount of mutagen and time of exposure may need to be empirically determined to give 30-40% survival. Survivals of 10% or less are to be avoided since the ungerminated spore population may be as high as 5%. The treated spores may be stored at 4° with little loss of viability for several days. Mutagenesis and Cloning with Transposon Tn917 An excellent set of vectors for transposon mutagenesis using Tn917 has been constructed by Youngman and colleagues 13-~5This system allows 12 G. Balassa, Mol. Gen. Genet. 104, 73 (1969). [3 p. j. Youngman, J. B. Perkins, and R. Losick, Proc. Natl. Acad. Sci. U.S.A. 80, 2305 (1983).
[13]
GENETIC ANALYSISIN Bacillus subtilis
319
isolation of transposition mutations with selection for Tn917 erythromycin resistance (Em R) by placing a strain with a temperature sensitive plasmid containing Tn917 at the nonpermissive temperature. The transposon-induced mutations are very stable, and the Em R marker can be used easily in transduction and transformation experiments. Cloning of the chromosomal regions adjacent to transposition-induced mutations is readily accomplished using a two-step procedure) 6 A pBR322 replicon containing a cat gene is first integrated into the transposon, and then the chromosomal DNA is digested with a restriction endonuclease, ligated, and transformed into E. coli as described above for recovery of integrative vectors. This system has proved to be very useful for cloning genes of interest from several chromosomal regions even though Tn917 insertions tend to cluster at the terminus of the chromosome, making the mutation search a little more arduous. The details of using Tn917 have been described) 7 Integrative Vectors for Studying Gene Expression One of the key techniques for studying gene expression in vivo is the coupling of the regulatory regions of agenc to the synthesis of/3galactosidase. This technique is particularly important in sporulation of B. subtilis, where determination of the timing of a gene's expression is one indication of its role in the temporal program of developmental transcription. For the identification of new genes that express their information under some controlled conditions, both transposons and integrative vectors may be used. Youngman and colleagues have developed Tn9I 7 derivatives that express/3-galactosidase when integrated into a transcribed region of the chromosome. Is If the transposon happens to be in agcne controlled by the conditions of interest, the mutant strain may be recovered by blue/white screening. This technique has been used to isolate several new sporulation-associated mutations.19 Integrative vectors for this purpose have been developed to generate either translation or transcription fusions to /3-galactosidase2° (also M. Perego and J. A. Hoch, unpublished data, 1988). These vectors are used by generating a random library of cloned fragments in the vector in vitro 14 j. B. Perkins and P. J. Youngman, Plasmid 12, 119 (1984). f5 p. j. Youngman, P. Zuber, J. B. Perkins, K. Sandman, M. Igo, and R. Losick, Science 228, 285 (1985). 16 p. Youngman, J. B. Perkins, and R. Losick, Mol. Gen. Genet. 195, 424 (1984). 17 p. Youngman, in "Plasmids: A Practical Approach" (K. Hardy, ed), p. 79. IRL Press, Oxford, 1986. is j. B. Perkins and P. J. Youngman, Proc. Natl. Acad. Sci. U.S.A. 83, 140 (1986). I9 K. Sandman, R. Losick, and P. Youngman, Genetics 117, 603 (1987). 20 F. A. Ferrari, K. Trach, and J. A. Hoch, J. Bacteriol. 161, 556 (1985).
320
OTHER BACTERIAL SYSTEMS
[13]
followed by transformation in E. coli. The pooled transformants are then transformed into B. subtilis and plated for Cm R with blue/white screening using the competence regimen. Integrative vectors have the advantage that the gene fused to fl-galactosidase may be unaltered in one-half of the duplications generated, and therefore genes whose inactivation would be lethal can be recovered as fusions. Transposons are easier to use, as no E. coli step is required, and they are more stable than integrative vectors, but they suffer from a lack of randomness and the vectors available cannot be used in temperature-sensitive strains. Fusion to fl-galactosidase in vitro is a mainstay in the study of cloned genes. Several integrative vectors with the capacity for generating translation or transcription fusions to fl-galactosidase have been described. These vectors integrate at the region of homology provided by the cloned fragment, which may not be desirable in some instances. In order to avoid this problem a/3-galactosidase fusion vector has been described that can be integrated into the amylase gene of B. subtilis with loss of amylase activity. 21 This vector, pDH32, places the/3-galactosidase fusion opposite to the direction of transcription of the amylase gene, and, because homology to amylase exists on both sides of the fusion and the CAT gene used for selection, the fusion integrates by a double crossover event in a stable configuration with loss of the vector sequences. This system has been used extensively for the study of expression of several genes, fl-Galactosidase fusions may also be placed in the ~b105 prophage to isolate them from the chromosomal region being studied. 22 A clever technique has been devised to transfer Tn917 fl-galactosidase fusions to the SPfl prophage. 23 The SPfl prophage may be induced to form a specialized transducing lysate which can be used to infect strains of interest.
21 H. Shimotsu and D. J. Henner, Gene 43, 85 (1986). 22 j. Errington, J. Gen. Microbiol. 132, 2953 (1986). N. Ramakrishna, E. Dubnau, and I. Smith, Nucleic Acids Res. 12, 1779 (1984).
GENETIC ANALYSISIN Bacillus subtilis
305
[13] G e n e t i c A n a l y s i s in Bacillus subtilis
By JAMES A. HOCH Introduction Of the bacteria Bacillus subtilis is the easiest organism to manipulate genetically. The armamentarium of genetic tools includes PBS 1 transduction which transfers very large [> 150-200 kilobase (kb)] fragments of the chromosome and DNA-mediated transformation which utilizes smaller DNA fragments (40 kb or less). The chromosomal location of new markers can be rapidly determined by transducing a set of auxotrophic strains covering all regions of the chromosome.l This transduction system takes the place of conjugation, which has never been developed for this organism. DNA-mediated transformation occurs at high frequency and is most useful for fine structure analysis of chromosomal regions up to 10 kb. In addition, a protoplast transformation system has been developed to allow easy transfer of plasmids between strains. Strategic Considerations In a common scenario, an investigator has isolated a series of mutants known to affect a particular pathway or phenomenon and wishes to determine the number of loci involved and their chromosomal locations. The first step in determining the chromosomal locations of the loci is to transduce a set of strains with auxotrophic markers conveniently located around the chromosome such that the entire chromosome will be covered.l Figure 1 shows the genetic markers that may be conveniently used for such an analysis. A PBS1 transducing lysate is prepared on the strain of interest and used to transduce each recipient to test for linkage. In practice all of the crosses can be carried out in a single day and a general chromosomal location determined in a week. Once the general location of the locus is obtained, further fine structure analysis of its location should be carried out by transformation. A large variety of genetic markers exist in virtually all regions of the chromosome. 2 Before embarking on genetic studies in B. subtilis, it is strongly suggested that the classic paper by C. Anagnostopoulos and co-workers 3 be 1 M. A. D e d o n d e r , J. A. L e p e s a n t , J. L e p e s a n t - K e j z l a r o v a , A. BiUault, M. Steinmetz, and F. K u n s t , Appl. Environ. Microbiol. 33, 989 (1977). 2 p. j. Piggot a n d J. A. H o c h , Microbiol. Rev. 49, 158 (1985). 3 M. Barat, C. A n a g n o s t o p o u l o s , a n d A . - M . Schneider, J. Bacteriol. 90, 357 (1965).
METHODS IN ENZYMOLOGY,VOL. 204
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
306
OTHER BACTERIAL SYSTEMS thiC
[13]
purAguaAcysA ~ a!ol
ctr~~''r 360/0~ " ~ 1
hisA~o,.
- "
~purB
thrB-~ '~Tn
Off ~..glyB
,roo . . . . . aroD ~
pyrA
trPCm'-tB ~ t K~, gltA glnA sw/~ FzG. 1. Landmark loci of the B. subtilis chromosome. The chromosome is divided by
degrees. Strains bearingthese markers are used to locateunknownmarkersby transduction. Strains are availablefromthe BacillusGeneticStock Center, Ohio State University,Columbus, Ohio.
read. This publication is a virtual treasure trove of genetic information and techniques and explores in detail techniques such as the recombination index that are not explained here.
Transformation and TransductionProcedures
Solutions PAB: 17.5 g antibiotic medium 3 (Difco, Detroit, MI) dissolved in 1 liter deionized water. Autoclave. TBAB: 17.5 g tryptose blood agar base (Difco) and 8.5 g Bacto Agate (Difco) suspended in 1 liter deionized water. Autoclave and dispense 25 ml in petri dishes. MG: 2 g ammonium sulfate [(NH4)2SO4]; 14 g potassium phosphate, dibasic (K2HPO4); 6 g potassium phosphate, monobasic (KH2PO4); 1 g sodium citrate (Na3C6HsO7 • 2H20); 0.2 g magnesium sulfate (MgSO4 • 7H20). Dissolve each of the ingredients successively in 1 liter of deionized water. Autoclave. Add 10 ml of sterile 50% glucose. For MG plates make a 2 × solution of salts and a 2 × solution of agar (Bacto-agar, Difco) to give 1.5% final agar concentration. Autoclave separately. Mix at 50° and add 10 ml of 50% glucose per liter. Fifty percent of glucose is easily made by dissolving 500 g glucose in 750 ml distilled water.
GENETIC ANALYSISIN Bacillus subtilis
[13]
307
CH: 5.0 g casein hydrolyzate, acid (Difco), dissolved in 100 ml deionized water. Autoclave. Note that CH does not contain tryptophan, glutamine, purines, pyrimidines, or vitamins. Amino acid stock solutions: 5 mg/ml in deionized water. Autoclave.
First Growth Period Supplemented MG. The final concentration of additives is 0.02% CH and 50 t~g/ml of any amino acid purine or pyrimidine required by the recipient. For one transformation: 1. Dilute 0.1 ml of CH in 2.4 ml of MG. Add 0.25 ml to a 20 × 150 mm culture tube. 2. Dilute 0.1 ml amino acid stock solution in 0.9 ml of MG. Add 0.25 ml of each amino acid required by the recipient to the above tube. Add sufficient MG to bring the volume to 2.5 ml.
Second Growth Period Supplemented MG. The final concentration of additives is 0.01% CH and 5/xg/ml of required amino acid. I. Make a 1/10 dilution of the required amino acid dilution and a I/2 dilution of CH dilution from First Growth ,Period Supplemented MG. 2. To a 20 x 150 mm culture tube add 0.1 ml of CH, 0.1 ml of each amino acid dilution, 0.1 ml 50 mM MgSO4, and MG to 0.9 ml.
Chromosomal DNA Preparation Inoculate the donor strain lightly in 20 ml of MG supplemented with 20 t~g/ml amino acid requirements and 0.05% CH in a 250-ml culture flask. Grow overnight with shaking at 37°. Centrifuge the culture I0 min at 4 ° at 7000 rpm (SS-34 head in a SorvaU centrifuge). Resuspend the pellet in 2 ml of TE (10 mM Tris, 1 mM Na2EDTA , pH 8.0) in a 16 × 125 mm culture tube and add 5 mg/ml fresh lysozyme and 8/xl of a solution of RNase at 10 mg/ml. Incubate for 30 min at 37°. Add proteinase K to 1 mg/ml and incubate for 15 min at 37 °. Next add 200/xl of 10% sodium dodecyl sulfate followed by 2 ml of phenol saturated with TE. Shake gently for 1 min. Centrifuge to separate the layers and extract the top layer with 2 ml phenol-chloroform (1 : 1) followed by 2 ml chloroform. Four milliliters of 95% (v/v) ethanol is layered on the final supernatant, and the D N A is spooled out on a fine glass rod or Pasteur pipette. Dissolve the DNA in 200/zl of TE.
Transformation Transformation requires the preparation of a cell culture competent to take up DNA. This mystical state occurs at the beginning of stationary phase and is associated with the onset of sporulation. Some mutants are difficult to bring to competence and therefore transform poorly. Many
308
OTHER BACTERIAL SYSTEMS
[13]
times this is a result of poor growth in the First Growth Period, and the simple addition of a little yeast extract (e.g., 0.01%) will solve the problem, Presented below is the original recipe for transformation devised by C. Anagnostopoulos and Spizizen4; which works well on a wide variety of strains.
First Growth Period 1. Streak the recipient strain on a TBAB plate the evening before the experiment. Incubate at 37 °. 2. From the fresh overnight TBAB culture sufficient cells are taken to give a lightly turbid suspension in 2.5 ml of First Growth Period Supplemented MG in a 20 x 150 mm sterile culture tube. 3. Incubate with shaking at 37° for 4 hr. The culture should be quite turbid; if not, incubate an additional 1 hr.
Second Growth Period 1. Prepare two culture tubes for the second period by diluting 0. I ml of culture in 0.9 ml Second Period growth medium in each of two 20 x 150 mm tubes. 2. To one tube add 0.1 ml of DNA solution (1 to 0.0001 /zg DNA) diluted in MG. To the second tube (the reversion control) only add MG. 3. Incubate with shaking at 37° for 90 min. 4. Prepare serial dilution tubes containing 0.9 ml MG and dilute the transformation tube 1/I0, 1/100, and 1/1000. Spread on separate MG plates 0.1 ml of the undiluted culture or 0.1 ml of each dilution along with 0.1 ml of appropriate amino acid stock solution required by the recipient but not selected for in this experiment. 5. Spread 0. I ml of undiluted reversion control culture on appropriately supplemented plates. 6. Incubate plates at 37° for 48 hr. This standard transformation procedure will yield around 100 colonies on the 1/I000 dilution plate for highly competent cells at DNA saturation. This corresponds to a frequency of 1% transformation. DNA saturation under these conditions is around 0.I-1.0 mg DNA/ml.
Protoplast Transformation The introduction of replicating plasmids into B. subtilis is most efficient with plasmid multimers. Furthermore, if the plasmid contains an insert of DNA homologous to the B. subtilis chromosome, it is likely to be subjected 4 C. Anagnostopoulos and J. Spizizen, J. Bacteriol. 81, 741 (1961).
[13]
GENETIC ANALYSISIN Bacillus subtilis
309
to a recombination event that can cause loss of the insert or gene conversion. Therefore, under these conditions it is best to introduce plasmids into recipients using the protoplast transformation procedure. Presented below is a simplified version (M. Zukowski, personal communication, 1990) of the original Chang and Cohen procedure: The procedure works well with minipreparation plasmid DNA. Solutions 2 × SMM buffer: 1.0 M sucrose, 40 mM sodium maleate, 40 mM MgCI 2 . Adjust to pH 6.5 and autoclave. SMMP medium: Mix together equal volumes of 4 x PAB (autoclaved) and 2 x SMM (autoclaved). PEG solution: 40 g polyethylene glycol (PEG) 6000, 50 ml of 2 x SMM buffer. Bring the volume to 100 ml with deionized water and autoclave. DM3 Regeneration Medium 1. Mix, in 700 ml deionized water, 135 g sodium succinate, 5 g casamino acids (Difco), 5 g yeast extract, 1.5 g KH~POa, and 3.5 g K2HPO4. 2. Adjust to pH 7.2. 3. Bring the volume to 950 ml with deionized water. 4. Add 8 g agar. 5. Autoclave. 6. Cool to approximately 55 °. 7. Add 25 ml of 20% sterile glucose (preheated to 55°). 8. Add 20 ml of 1 M MgCI2 (preheated to 55°). 9. Add 5 ml of 2% bovine serum albumin (BSA, freshly prepared, filter-sterilized). 10. Add antibiotics as required: chloramphenicol, 10 tzg/ml final concentration; kanamycin, 100/xg/ml final concentration. 11. Pour plates, leave on the benchtop overnight, then store at 4 ° for no longer than 1 month. Procedure 1. A starter culture is grown overnight in 10 ml of PAB at 37° with shaking. 2. The following morning, 0.5 ml of starter culture is inoculated into 50 ml of PAB. Shake the culture at 37° until the cell mass reaches an OD660 of 0.4-0.5. 3. Transfer the culture into a 50-ml plastic centrifuge tube. Pellet the s S. Chang and S. N. Cohen, Mol. Gen. Genet. 168, 111 (1979).
310
OTHER BACTERIAL SYSTEMS
[13]
cells by centrifugation (e.g., I0 min at 6000 rpm in Beckman TJ-6 centrifuge with a fixed-angle rotor). 4. Resuspend the pellet in 5 ml of SMMP with 2 mg/ml lysozyme freshly added. The solution may be filter-sterilized before adding to cells. 5. Incubate the cells in a plastic centrifuge tube at 37° with gentle rocking (about 100 rpm) for 90 min (60 rain may be satisfactory in some cases). Microscopic evaluation of protoplasting efficiency may be called for, but loss of schlieren in the suspension should be indicative. 6. Pellet protoplasts by centrifugation (e.g., 10 min at 5000 rpm at 4° in Beckman TJ-6 centrifuge with a fixed-angle rotor). 7. Pour off the supernatant and add 2.5 ml of SMMP. Resuspend the protoplasts by vigorous agitation of the tube with fingers. Do not use a vortex.
8. a. Mix the plasmid DNA with an equal volume of 2 × SMM in a 50-ml plastic centrifuge tube. b. Add 0.5 ml of the protoplast suspension. Agitate the tube lightly with fingers. c. Add 1.5 ml of the PEG solution. Gently mix by rolling the tube manually. d. After 2 rain of exposure to the PEG, add 5 ml SMMP. 9. Repeat Step 6. I0. Resuspend the pellet in I ml of SMMP; incubate at 37° with gentle shaking for 90 min. 11. Plate 0.1-ml samples onto fresh DM3 regeneration medium plates to which antibiotics have been added as required. Incubate plates at 37° for 48-72 hr. Expect over 105 transformants//Lg for uncut DNA and 104-10 5 transformants//.~g for cut and ligated DNA. Expect no transformants if MgC12 was omitted from solutions.
PBS1 Transduction PBS I is a large transducing phage with the capability of encapsulating 150-200 kb of chromosomal DNA in the transducing particle. It depends on motile flagella for attachment and therefore is not useful for Fla-, Mot-, or any other strain with mutations that impair motility. The vast majority of researchers that use it have never seen a PBS1 plaque since it is an exceedingly poor plaque former. Despite these problems this transducing system works well for locating markers on the chromosome. PBS1 Stock Lysate. PBS1 stock lysates are prepared on the highly motile Bacillus licheniformis 8480 strain or any highly motile B. subtilis strain. Bacillus licheniformis 8480 (strain 5A1 in the Bacillus Genetic Stock Center Collection) cells from an overnight TBAB plate are inoculated at light turbidity into 2.5 ml of PAB in a 20 x 150 mm culture tube. Shake
[13]
GENETIC ANALYSISIN Bacillus subtilis
311
the tube at 37° for 2.5 hr. Add 0.1 ml of PBS1 stock lysate; allow adsorption for 5 min at room temperature. Dilute the tube into 25 ml of PAB in a 250ml culture flask. Shake at 37° for 2 hr. Place the flask in 37° incubator overnight without shaking. Centrifuge the lysate at 10,000 g for 20 min at 4° to pellet debris. Add 50/zg/ml DNase I (dissolved in 0.01 M MgSOa) and incubate 30 min at room temperature. Filter-sterilize the supernatant through a 0.45-/~m filter. Store at 4°. The lysate will be stable for at least 1 year. Preparation of Donor Lysates. The donor strain is streaked on a TBAB plate and grown overnight at 37°. Donor cells are inoculated at light turbidity (OD540 -0.1) into 2.5 ml of PAB in a 20 × 150 mm culture tube. Shake at 37° for 2.5 hr. The cells at this time are just beginning to become motile. Add 0.1 ml of the PBS 1 stock lysate. Incubate at room temperature for 5 min. Dilute into 25 ml of PAB in a 250-ml culture flask. Incubate with shaking at 37° for 1 hr. Add chloramphenicol to 5/~g/ml (spectinomycin or no antibiotic at all may be used if the strain is CmR). Shake for an additional 2 hr at 37°. Let the culture stand without shaking at 37° overnight. Centrifuge the lysate at 10,000 g for 20 min at 4° to remove debris. Add 50/~g/ml DNase I and incubate at room temperature for 30 min. Filtersterilize through a 0.45/.tm filter. Store the lysate at 4°. Although this procedure seems empirical, and is, it works almost every time, and after several times it seems natural. It is important to treat the lysate with DNase I because it will not filter well without it, and this step avoids transformation of the recipients. Transduction Procedure. The recipient strain is grown overnight on a TBAB plate at 37° and inoculated at light turbidity into 2.5 ml of PAB in a 20 x 120 mm culture tube. Shake at 37° for 4 hr. The cells at this stage should be motile and uniform in size when examined microscopically. Add 0.5 ml of cells and 0.5 ml of donor lysate to a 16 × 125 mm culture tube. To a second tube add 0.5 ml cells and 0.5 ml of PAB for a reversion control. Shake both tubes for 20 min at 37°. Plate 0. l ml of transduction and reversion cultures on appropriately supplemented MG plates for selection of recipient markers. This procedure normally gives between 50 and 500 transductants on the selection plate. Since 0.1 ml of rich medium is plated on the MG plates a slight lawn appears on the plate. This can be a problem with certain markers such as vitamin deficiencies, and, if so, the transduced cells can be centrifuged and washed with MG before plating. Analysis of Genetic Data The basic measure relating genetic and physical distance between two markers in transduction and transformation is the recombination unit. This is defined as percent recombination = 100 (1 - cotransfer), where
312
OTHER BACTERIAL SYSTEMS
[13]
degrees
1.0
5.5
\
I1.0
16.6
0:4
016
22.1
27.6
i
0.8 0.6 C
\,
\
x\
0.4
x~
0.2 X""--
012
/
X...._ X ~
0.8
| -
I.(3
FIG. 2. Relationship between cotransfer values in transduction and physical distance between markers on the chromosome. A graph of the Kemper formula C = (1 - t) + t(ln t) relates cotransfer values to the degrees separating two markers on the chromosome. 5,6 C is the cotransfer value between markers, and t is the length of the PBSI transducing DNA fragment.
cotransfer of linked markers is the number of double transformants divided by the single transformants. Markers further apart on the chromosome will have a smaller cotransfer value and a larger recombination value than closely linked markers. To be meaningful in terms of the physical distance separating two markers, the recombination values can be converted to physical distance using the Kemper formula, C = (1 - t) + t(ln t), where C is the cotransfer value and t is the fractional length of the donor DNA separating the two markers. 6 If the average size of the donor DNA is known (T), the physical distance between the two markers (D) can be calculated from D = tT. F i g u r e 2 7 shows the Kemper formula in graphical form where the t value in PBS I transduction has been related (approximately) to the degrees separating the markers on the chromosome. The graph makes it clear that cotransfer or recombination values are not additive. For example, a marker, X, located equidistant between two outside markers, A and B, and showing 50% recombination to each would be about 0.2t or 5.5 ° from each. The recombination values expected between A and B markers would not be 100% (50% + 50%) but rather 75% (0.2t + 0.20. The assumptions and calculations behind this figure have been detailed elsewhere. 7 The Kemper analysis is most useful in placing markers on the chromosome 6 j. Kemper, J. Bacteriol. 117, 94 (1974). 7 D. J. Henner and J. A. Hoch, Microbiol. Rev. 44, 57 (1980).
[13]
GENETIC ANALYSISIN Bacillus subtilis
313
5.00
i
15
4.25
3.50
u)
2.75
0 .J
2.00 -4
I
I
I
I
-3
-2
-1
0
Log ~
0 1
{]t.Q/ml)
FIG. 3. Relationshipbetween singleand double transformationand DNA concentration in transformationof weaklylinked markers. The data (fromRef. 2) showthe Leu÷ (©) and Phe÷ Leu÷ (A) transformantsfor the weaklylinked markersphe-1 and leu-1.
map by PBS1 transduction where the size of the transducing fragment is thought to be uniform. In transformation, the size of the transforming DNA can vary greatly from preparation to preparation, and the values for C are size dependent, particularly the lower values. In general 7-10% recombination between two markers in transformation means that about 1 kb of DNA separates the two markers. The interested reader is referred to Ref. 3 for detailed examples of linkage analysis using both systems and to Henner and Hoch 6 for an in-depth study of the application of the Kemper formula, Congression and Strain Construction Congression is a phenomenon unique to transformation and refers to the uptake of more than one transforming DNA fragment by a competent cell, particularly at high DNA concentrations. Congression is a two-edged sword in that it disturbs linkage analysis of distant markers but is extremely helpful in strain construction. Figure 3 shows the effect of congression on the weakly linked phe and leu markersJ As the DNA concentration reaches saturating levels for the competent cell population, the number of double transformants Phe + Leu + increases greatly in the cross wild-type
314
OTHER BACTERIAL SYSTEMS
[13]
DNA x phe leu recipient. The recombination values between closely linked markers are not subject to this effect. The example shown in Fig. 3 is of weakly linked markers where the recombination value stabilizes at nonsaturating DNA concentrations. Completely unlinked markers show no cotransfer at nonsaturating DNA concentrations. Congression is the major technique used for strain construction if unlinked markers are involved. Any selectable marker in the recipient can be exchanged for one in the donor by transformation using DNA concentrations greater than 1/xg/ml. For example, a trpC2 leu-1 recipient could be changed to a phe-1 leu-1 strain by transforming with DNA containing the phe-I allele with selection for Trp ÷ and searching among the Trp + transformants for Phe- recombinants. Piggot and de Lencastre have described a method to generate multiple marked strains where the recipient does not lose the selected marker. 8 This method is based on the observation that the products of certain early sporulation genes, for instance, spolIA, are required only in the mother cell and not in the developing forespore. Thus, if one transforms a spolIA recipient for Spo ÷ with wild-type DNA and selects for Spo ÷, some of the transformants will be genotypically spolIA because the transforming Spo ÷ DNA integrated into the chromosome that remained in the mother cell and, therefore, allowed sporulation to occur but did not convert the forespore chromosome to Spo ÷. In practice, one can transform a spolIA recipient with saturating phe-1 DNA, for example, selecting Spo + and searching for phe-1 spolIA recombinants among the transformants. Successive markers can then be added to this strain by repeating the procedure with new donor DNA. Thus, congression is a powerful tool to facilitate strain construction with characterized markers. It is also a simple and important means to backcross primary mutant strains to determine if all the phenotypes of a mutant can be ascribed to a single mutation.
Studies of Complementation and Dominance Using Integrative Vectors Since both complementation and dominance may reflect the subtle interplay of regulatory elements, the vectors to study these phenomena were designed to balance the concentration of the elements involved. The majority of these studies utilize integrative vectors which are basically Escherichia coli plasmids carrying an antibiotic resistance gene that is expressed in B. subtilis. One of the simplest and most utilized of these vectors is pJH101, which has a CAT (chloramphenicol acetyltransferase) g P. J. Piggot and H. de Lencastre, J. Gen. Microbiol. 106, 191 (1978).
[13]
GENETICANALYSISIN Bacillus subtilis
315
NdeI-Sau 3A)
Xm°,,,f..;-
~,~~,,~j~,(NdeI-Hpa
II)
Hind III c/orang site
FIG. 4. Map of the integrative vector pJMl03. The vector was constructed by ligating a filled-in H p a l I - S a u 3 A fragment of pC194 IS. Horinouchi and B. Weisblum, J. Bacteriol. 150, 815 (1982)] into the NdeI site ofpUC19. A sister plasmid (pJMl02) was also constructed from pUCI8 (M. Perego and J. A. Hoch, unpublished, 1988).
gene in the PoulI site of pBR322. 9 Integrative vectors are incapable of replication in B. subtilis and therefore do not transform B. subtilis to chloramphenicol resistance. If a region of homology to the chromosome is cloned on the vector, transformation results in chloramphenicol-resistant colonies which are so-called Campbell recombinants where the inserted vector is flanked by a duplication of the original chromosome fragment carried on the vector. As long as this fragment is big enough to prevent interruption of the transcription unit being studied, meaningful complementation and dominance results between alleles can be obtained since there are only two copies of the region being studied on the chromosome. The vector is also quite stable when integrated. Figure 4 shows a newer integrative vector, pJM103, which has a CAT gene in pUC19. Kanamycin-resistant derivatives of pUC18 and pUC19 are also available (M. Perego and J. A. Hoch, unpublished, 1988). pJM103 has all the properties of pUC19 in E. coli, therefore cloning of B. subtilis fragments may be carded out in E. coli utilizing the blue/white Lac selection system. After cloning and characterization of the DNA fragment of interest, the plasmid preparation is used to transform B. subtilis using the competence regimen outlined above. Minipreparations of the plasmid work very well. 1° Chloramphenicol resistance (CmR) may be selected on 9 F. A. Ferrari, A. Nguyen, D. Lang, and J. A. Hoch, J. Bacteriol. 154, 1513 (1983). i0 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979).
316
OTHER BACTERIAL SYSTEMS
[13]
any enriched medium such as TBAB containing 5/xg/ml chloramphenicol. Cm R transformants are readily obtained with DNA inserts as small as 400-500 base pairs (bp), but the yield falls rapidly below this size range. The smallest DNA insert that we have found to give a Campbell recombinant is 149 bp. Integrated vectors may be treated as a locus in transformation and transduction studies. PBS 1 transduction may be used to locate the vector on the chromosome. In transformation studies the vector plus duplication is a significant fraction of the transforming DNA segment, and therefore larger DNA fragments (i.e., gently prepared DNA) are required to show linkage to closely linked markers. Recombination values are depressed because of the longer DNA fragments, and severe recombinational polarity is observed. Polarity results from the fact that any shearing within the vector in the DNA preparation results in loss of the Cm R recombinant. Because of these complications, recombination values in transformation are not a reliable indicator of the location of integrated plasmids. A plasmid system for complementation and dominance studies has been constructed to allow integration in the SP/3 prophage found in all B. subtilis strains. II This plasmid, pFH7, contains a region of homology to SP/3 and integrates into it on transformation in B. subtilis. The advantages of this system are that the gene of interest is inserted in another location in the chromosome, without disturbing its normal location, and induction of the prophage results in a specialized transducing phage carrying the gene. Mutagenesis and Cloning with Integrative Vectors Integrative vectors are highly mutagenic if the insert chromosomal DNA fragments are wholly contained within a gene or transcription unit. Both copies of the gene or transcription unit generated by the integration event are disrupted in this case. Insert fragments that stop within a gene or transcription unit and extend beyond either of its ends are nonmutagenic since one good copy of the gene is generated in the integration event. In a random mutagenesis experiment using TaqI chromosomal fragments cloned in the AccI site of pJM102, 1% auxotrophs and 10% sporulation mutants were obtained among the Cm R transformants (M. Perego, unpublished data, 1989). Mutations generated in this fashion allow the affected locus to be easily cloned by excision of the vector and adjacent sequences with restriction enzymes, ligation in vitro, and transformation in E. coli. The procedure for recovery of integrated plasmids is straightforward. 11 E. Ferrari and J. A. Hoch, Mol. Gen. Genet. 189, 321 (1983),
[13]
GENETIC ANALYSIS IN Bacillus subtilis
Hindlll
I
EcoRI
H
Hlndlll
317
EcoRI
I I
E
I
FIG. 5. Integration of pJM103 in the chromosome. The hypothetical genes labeled A - F are adjacent to the cloned fragment X-Y. The fragments H and E are the expected plasmids recovered by HindlII and EcoRI digestions, respectively.
An overnight culture of the strain containing the integrated vector is used to prepare chromosomal DNA as described above. Two micrograms of this DNA is digested with the appropriate restriction endonuclease in 30 /.d of the appropriate buffer for the enzyme used, and 1/xg is removed to check the digestion on an agarose gel. The remaining 15 t~l is diluted to 100 /zl with deionized water and extracted with phenol followed by a chloroform extraction. The aqueous layer (-80/zl) is diluted with ligation buffer to 100/zl, ligase (1 unit) is added, and the mixture is incubated at 15° overnight. The entire ligation mixture is used to transform E. coli DH5a with selection for ampiciUin resistance. Usually 20-50 transformants are obtained. Figure 5 shows an example of the insertion of pJM 103 into a cluster of genes and the expected plasmids that would be obtained by excision with EcoRI (E) or HindlII (H). Any restriction enzyme with a site within the multilinker ofpJM103 can be used, but the direction of walking will depend on the orientation of the site to the inserted fragment. Gene disruption experiments using integrative vectors are standard procedure for the analysis of cloned DNA fragments where the phenotype of a disrupted open reading frame of unknown function is in question. Mutagenesis by integrative vectors compares favorably in frequency with the best classic mutagenesis procedures. It can be used in a random procedure using total chromosomal DNA or directed specifically to a
318
OTHER BACTERIAL SYSTEMS
[13]
cloned region or gene. The locus of the mutagenic plasmid can be readily cloned from the affected strain. Mutation with Chemical Agents
Bacillus subtilis is susceptible to all the chemical mutagenic agents used for induction of mutations in other bacteria. However, a very effective method of mutagenesis unique to spore formers was described by Balassa.12 If spores are germinated in the presence of chloramphenicol to prevent protein synthesis, they are extremely susceptible to N-methyl-N'nitro-N-nitrosoguanidine mutagenesis. Up to 25% sporulation mutants and 5% auxotrophs were found among the survivors of such treatment. This procedure is probably applicable to any sporulating bacterium and to a variety of mutagenic agents. Procedure. A suspension of spores of the strain of interest is prepared in a sporulation medium such as AK agar (BBL, Cockeysville, MD) by streaking the strain on the agar plate, incubating at 37° for 48 hr, and harvesting the spores by scraping the plate with 5 ml of PAB. Vegetative cells are killed by the addition of a drop of chloroform, and the suspension is stored at 4 °. Fifty milliliters of PAB containing I00/~g/ml chloramphenicol in a 250 to 500-ml culture flask is inoculated with the spore suspension to give 107- l0 s spores/ml. The suspension is incubated with shaking at 37° for 1 hr to give 90-99% germination. Germinated spores turn phase-dark in a phase microscope in contrast to the highly refractile ungerminated spores. N-Methyl-N'-nitro-N-nitrosoguanidine is added to a final concentration of 50/zg/rnl, and incubation is continued for 30 rain. The suspension is then centrifuged 6000 g for 15 rain at 4 ° to pellet the spores, and the treated spores are resuspended in sterile MG and stored at 4 °. Dilutions of the treated spores are plated on the desired medium for mutant selection or screening. This procedure is extremely effective when used correctly. The amount of mutagen and time of exposure may need to be empirically determined to give 30-40% survival. Survivals of 10% or less are to be avoided since the ungerminated spore population may be as high as 5%. The treated spores may be stored at 4° with little loss of viability for several days. Mutagenesis and Cloning with Transposon Tn917 An excellent set of vectors for transposon mutagenesis using Tn917 has been constructed by Youngman and colleagues 13-~5This system allows 12 G. Balassa, Mol. Gen. Genet. 104, 73 (1969). [3 p. j. Youngman, J. B. Perkins, and R. Losick, Proc. Natl. Acad. Sci. U.S.A. 80, 2305 (1983).
[13]
GENETIC ANALYSISIN Bacillus subtilis
319
isolation of transposition mutations with selection for Tn917 erythromycin resistance (Em R) by placing a strain with a temperature sensitive plasmid containing Tn917 at the nonpermissive temperature. The transposon-induced mutations are very stable, and the Em R marker can be used easily in transduction and transformation experiments. Cloning of the chromosomal regions adjacent to transposition-induced mutations is readily accomplished using a two-step procedure) 6 A pBR322 replicon containing a cat gene is first integrated into the transposon, and then the chromosomal DNA is digested with a restriction endonuclease, ligated, and transformed into E. coli as described above for recovery of integrative vectors. This system has proved to be very useful for cloning genes of interest from several chromosomal regions even though Tn917 insertions tend to cluster at the terminus of the chromosome, making the mutation search a little more arduous. The details of using Tn917 have been described) 7 Integrative Vectors for Studying Gene Expression One of the key techniques for studying gene expression in vivo is the coupling of the regulatory regions of agenc to the synthesis of/3galactosidase. This technique is particularly important in sporulation of B. subtilis, where determination of the timing of a gene's expression is one indication of its role in the temporal program of developmental transcription. For the identification of new genes that express their information under some controlled conditions, both transposons and integrative vectors may be used. Youngman and colleagues have developed Tn9I 7 derivatives that express/3-galactosidase when integrated into a transcribed region of the chromosome. Is If the transposon happens to be in agcne controlled by the conditions of interest, the mutant strain may be recovered by blue/white screening. This technique has been used to isolate several new sporulation-associated mutations.19 Integrative vectors for this purpose have been developed to generate either translation or transcription fusions to /3-galactosidase2° (also M. Perego and J. A. Hoch, unpublished data, 1988). These vectors are used by generating a random library of cloned fragments in the vector in vitro 14 j. B. Perkins and P. J. Youngman, Plasmid 12, 119 (1984). f5 p. j. Youngman, P. Zuber, J. B. Perkins, K. Sandman, M. Igo, and R. Losick, Science 228, 285 (1985). 16 p. Youngman, J. B. Perkins, and R. Losick, Mol. Gen. Genet. 195, 424 (1984). 17 p. Youngman, in "Plasmids: A Practical Approach" (K. Hardy, ed), p. 79. IRL Press, Oxford, 1986. is j. B. Perkins and P. J. Youngman, Proc. Natl. Acad. Sci. U.S.A. 83, 140 (1986). I9 K. Sandman, R. Losick, and P. Youngman, Genetics 117, 603 (1987). 20 F. A. Ferrari, K. Trach, and J. A. Hoch, J. Bacteriol. 161, 556 (1985).
320
OTHER BACTERIAL SYSTEMS
[13]
followed by transformation in E. coli. The pooled transformants are then transformed into B. subtilis and plated for Cm R with blue/white screening using the competence regimen. Integrative vectors have the advantage that the gene fused to fl-galactosidase may be unaltered in one-half of the duplications generated, and therefore genes whose inactivation would be lethal can be recovered as fusions. Transposons are easier to use, as no E. coli step is required, and they are more stable than integrative vectors, but they suffer from a lack of randomness and the vectors available cannot be used in temperature-sensitive strains. Fusion to fl-galactosidase in vitro is a mainstay in the study of cloned genes. Several integrative vectors with the capacity for generating translation or transcription fusions to fl-galactosidase have been described. These vectors integrate at the region of homology provided by the cloned fragment, which may not be desirable in some instances. In order to avoid this problem a/3-galactosidase fusion vector has been described that can be integrated into the amylase gene of B. subtilis with loss of amylase activity. 21 This vector, pDH32, places the/3-galactosidase fusion opposite to the direction of transcription of the amylase gene, and, because homology to amylase exists on both sides of the fusion and the CAT gene used for selection, the fusion integrates by a double crossover event in a stable configuration with loss of the vector sequences. This system has been used extensively for the study of expression of several genes, fl-Galactosidase fusions may also be placed in the ~b105 prophage to isolate them from the chromosomal region being studied. 22 A clever technique has been devised to transfer Tn917 fl-galactosidase fusions to the SPfl prophage. 23 The SPfl prophage may be induced to form a specialized transducing lysate which can be used to infect strains of interest.
21 H. Shimotsu and D. J. Henner, Gene 43, 85 (1986). 22 j. Errington, J. Gen. Microbiol. 132, 2953 (1986). N. Ramakrishna, E. Dubnau, and I. Smith, Nucleic Acids Res. 12, 1779 (1984).
