GENETIC TRANSFORMATION OF Azospi ri 1 1 um brasilense BY PLASMID DNA
BY
ELZA MACHADO MENEZES
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981 ACKNOWLEDGMENTS
My special thanks go to Dr. Dennis E. Duggan, chairman of my graduate committee, for his unlimited patience in trying to under- stand my poor English and for his contributions in terms of discussions and suggestions.
My thanks go also to Dr. John T. Mullins and Dr. Phillip M.
Achey, the other members of my graduate committee, for their discus- sions and contributions in the course of this research.
I would like to acknowledge the financial support given to me by Universidade Federal Rural do Rio de Janeiro (UFRRJ), Programa de
Ensino Agricola Superior (PEAS), and Coordenadoria e Aperfeigoamento de Professores de Ensino Superior (CAPES).
Also my special thanks and acknowledgment go to Euri pedes, my husband, whose love, patience, and encouragement helped me through the duration of this research.
My thanks go to all the people who directly or indirectly helped me to get this work completed. TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS 11
iv LIST OF TABLES
ABSTRACT VI
INTRODUCTION 1
LITERATURE REVIEW
Transformation of Gram-Negative Bacteria ...... 2 Transformation of Gram-Positive Bacteria 6
MATERIALS AND METHODS
16 RESULTS
Isolation of Phage ^ Conjugation Transformation
DISCUSSION • 42
. 50 REFERENCES CITED . .
54 BIOGRAPHICAL SKETCH . LIST OF TABLES
Table Page
1 Bacterial strains used 10 6
2 Frequency of transconjugants using different donor and recipient strains 17
3 Transformation of Azospiri 1 1 urn with DNA from plasmid band separated by dye buoyant density gradient centrifugation 19
4 Effect of age of cells and incubation time with DNA on transformation frequency 20
5 Effect of cold phase time variation on transformation + frequency of ade 22
Effect of pH of DNA incubation buffer on transformation frequency of 1eu + 23
7 Effect of time of heat shock on transformation frequency 25 ++ 8 Effect of MN concentration on transformation frequency 26
9 Effect of DNA concentration on transformation frequency 27
10 Effect of shearing plasmid DNA on transformation frequency 29
11 Transformation of biosyntehtic genes with electrodialysed DNA from excised gel segments after electrophoresis 30
12 Transformation of biosynthetic genes with electrodialysed DNA from excised gel segments after electrophoresis 31
13 Transformation of biosynthetic genes with electrodialysed DNA from excised gel segments after elecgrophoresis 32
iv LIST OF TABLES (Continued)
Table Page
14 Transformation with DNA removed from crushed gel segments excised after electrophoresis 33
15 Transformation with DNA removed from crushed gel segments excised after el ectrophoresi s 34
16 Transformation with heterologous DNA from crushed gel segments excised after electrophoresis 36
17 Transformation of Azospiri 1 1 urn with homologous and with heterologous DNA 39
18 Transformation of Azospiri 11 urn with homologous and with heterologous DNA 40
19 Transformation of Azospirillum with homologous and with heterologous DNA 41
v i
Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
GENETIC TRANSFORMATION OF Azospirillum brasilense BY PLASMID DNA
By
Elza Machado Menezes
March 1981
Chairman: Dennis E. Duggan Major Department: Microbiology and Cell Science
A method for genetic transformation of E. col has been found to be useful for transformation of Azospirillum . Optimal conditions were found to be as follows: 1) six- hour- old cells; 2) 30 minutes of incubation of cells with DNA solution in the cold in the presence of
of heat shock, and 5) 100 mM MnCl and CaCl ; 3) pH 5.6; 4) 2.5 minutes 2 2 transformation MnCl in the presence of CaCl - We also found that trp 2 2 frequency increased with DNA concentration up to 30 yg/ml where this frequency was approximately 2xl0^/yg DNA. Shearing of CCC DNA isolated by dye buoyant density centrifugation reduced transformation frequency by nearly 2 log cycles.
Plasmid DNA from wild type donors, separated by dye buoyant density centrifugation, was found capable of transforming auxotrophic recipients to prototrophy. Similarly plasmid DNA from antibiotic resis- tant donors could transform sensitive recipients to resistance.
vi Plasmids were resolved into discrete bands by a sensitive gel electrophoresis technique. Supernatants from excised gel bands were electrodialyzed or crushed and used for transformation. Some of those bands transform for biosynthetic and antibiotic resistance markers.
Inter strain and inter species transformation frequencies were not lower than those of homologous matings. This suggests the absence of restriction mechanisms. INTRODUCTION
Azospirillum is an asymbiotic nitrogen-fixing bacterium currently
under investigation for its beneficial effects on grasses (Van Berkum
other plants (Kumari et al . , 1976). and Bohlool , 1980), cereals, and
a These bacteria fix nitrogen under mi croaerophi 1 ic conditions using
variety of carbon sources. Nitrogen fixation has also been reported
on the roots and in the rhizosphere of tropical grass (Dobereiner and
Day, 1976), maize (Von Bulow and Dobereiner, 1975), and rice (Diem
et al (1980), et al., 1978), when this organism is present. Umali-Garcia .
investigated the association between grass roots and Azospi ri 1 1 um
brasilense Sp 7. They observed that inoculated roots of pearl millet
and and guinea grass produced more mucilaginous sheath, root hairs,
lateral roots than did uninoculated sterile controls. Azospi ri Hum
species also produce phytohormones and have been shown to alter root
et al 1979). morphology in a manner similar to purified hormones (Tien . ,
Very little genetic information has been "extracted" from these
newly studied organisms partially because mutant strains have not been
available. The nif'mutants have been particularly difficult to isolate.
We have made a variety of mutants to be used in the development
study of the of a system of gene transfer which will be useful in the
genetics and the molecular biology of Azospi ri 1 1 um .
1 , , , a
LITERATURE REVIEW
The ability to introduce individual molecules of plasmids DNA
into bacterial cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the develop- ment of DNA-cloning methods.
Genetic transformation has proved to be one of the valuable techniques in studying genetic fine structures of several microorganisms.
Bacterial transformation using plasmid DNA has been reported in sev- eral different species of bacteria, including Diplococcus pneumoniae
(Cohen et al . 1972), Salmonella (Lacks et al . 1 974), E. coli
(Gryczan typhimuri urn (Lederberg and Cohen, 1974), Staphylococcus aureus
subtilis (Contente and Dubnau , 1979), et al . 1 978) , Bacillus
Nei sseri Haemophilus parai nfl uenzae (Gromkova and Goodgal , 1979),
qonorrhoeae (Sparling, 1966), and others.
Transformation of Gram-Negative Bacteria
Khan and Sen (1967) investigated the intraspecific and inter-
and valine specific transformation in Pseudomonas . Two isoleucine
requiring mutants of Pseudomonas aeruginosa had been transformed to
prototrophy using chromosomal DNA.
Kloos (1969) studied the factors that affect the transformation
of Micrococcus lysodei kti cus and he verified that the number of trans-
formants increased up to a saturation level of about 1 pg/ml DNA.
2 i i i
3
The transformation reached a maximal value after a 30 to 40 minute expo-
sure to DNA.
al studied the transformation to multiple anti- Cohen et . (1972)
biotic resistance by purified R-factor DNA in E. col cells treated with
CaCl . They showed that covalently-closed, catenated or open circular 2
forms of R-factor DNA are all effective in transformation, but denatur-
ation and sonication abolish the transforming ability of R-factor DNA
in this system.
Cosloy and Oishi (1973a, b) studied the nature of the transfor-
mation process in £. col by investigating various factors which affect
the efficiency of transformation. They found that CaCl^ treatment of
the recipient cells is absolutely necessary for transformation, the
efficiency of transformation is dependent upon temperature during incu-
bation of the recipient cells with DNA and the efficiency is also
affected by the molecular weight of the donor DNA used.
Lederberg and Cohen (1974) studied the transformation system of
Salmonella typhimurium by plasmid DNA and they found that pretreatment
of cells with MgCl before CaCl was necessary to obtain transformation 2 2
frequency as high as _E. col i .
Chakrabarty et al. (1975) developed a transformation system with
plasmid DNA and they described the conditions optimal for the transfor- mation of Pseudomonas putida and Escherichia coli with a drug-resi stance
factor (RP1) DNA, which specified resistance to carbeni ci 1 1 i n , tetra-
cycline, kanamycin, and neomycin.
Page and Sadoff (1976) studied the physioloical factors that
affect the transformation of Azotobacter vi nelandi and they found that 4
exogenous DNA towards the cells of the bacterium can be transformed by
very low frequencies end of exponential growth. Transformation occurs at
carried out in when the DNA is purified or when the transformation is
and at 30°C, liquid medium. Transformation is optimal at pH 7.0 to 7.1 polymer conditions which also coincide with minimal extracellular production.
of Sano and Kageyama (1977) reported the transformation retained plasmid DNA (RP ). The transformants Pseudomonas aeruginosa by 4
in the parent plasmid. all the drug-resistance characteristics present
were successful in transforming a plasmid- Holsters et al . (1978)
with the octopine Ti- free Agrobacteri urn with the P-type plasmid RP^,
nopaline Ti-plasmid TiC using plasmids TiB S and T i ACH and with the 5g 6 3 ^ et al (1972). These the freeze-thaw method described by Dityatkin . penetrate authors suggest that with this technique, the DNA molecules
through temporary into the frozen-thawed cells by passive diffusion
lesions in the cell wall and membrane.
Dagert and Ehrlich (1979) studied the competence of E. c.oJl
They found that cells to prolonged incubation in calcium chloride.
transformable and 20-30 times more E. col i cells are 4-6 times more
than immedi- competent after 24 h incubation in cold calcium chloride
x 10 ately after chi oride treatment. With 24 h competent cells, 2
transformants/pg of pBR 322 DNA were routinely obtained and over
20% of viable cells were transformed.
Gromkova and Goodgal (1979) described an efficient procedure
DMA. The for transformation of Haemophilus influenzae by plasmid
of competent cells procedure involved a new technique for preparation i i i
5
that increased the transforming efficiency of a plasmid amja about
100-fold. They grew the cells in brain heart infusion medium (BHI)
at 30°C without shaking. They added to the competent cells the DNA
solution plus BHI broth supplemented with NAD.
reported transformation at very low level. Mishra et al . (1979),
They transformed for streptomycin, novobiocin, and Kanamycin resistance
i n Azospi ri 1 1 urn brasi 1 ense Sp 7.
Page and Von Tigerstrom (1979) studied the optimum conditions
for transformation of Azotobacter vi nel andi i . They found that the opti-
mal transformation of A. vi nel andi OP required a 20-minute incubation of
the competent cells with DNA at 30°C in buffer (pH 6.0 to 8.0), con-
taining 8 mM magnesium sulfate.
Gill, Alexander, and Curtiss III (personal communication) used
the low pH method for transformation in £. col . They obtained best ++ ++ ++ results with either Mn or Mg and Ca treatments.
Oishi and Irbe (1977) consider the following possibilities to
explain the mechanism by which Ca Cl treatment facilitates DNA transport 2
through the _E. col membrane, increasing the transformation efficiency.
(1) Calcium ions neutralize the negative charge of the cell surfaces,
thus removing a barrier and providing the donor DNA with easier access
to all membranes; (2) Calcium ions denature a particular type of protein
in the membrane, thus affecting the penetration of DNA, and (3) Calcium
ions activate a latent enzyme which modifies the membrane structure to make it permeable to DNA. 6
Transformation of Gram-Positive Bacteria
Tomasz (1966) studied the mechanism responsible for controlling
the expression of competent state in Pneumococcus culture and he found
that the most important parameter affecting the time course of the com-
petence was the cell concentration. Constant high levels of competence
could be maintained in cultures growing in a continuous-dilution device.
Lacks et al. (1974) studied the role of a deoxyribonuclease in
the genetic transformation of Diplococcus pneumoniae . They found that
the entry of the DNA into the cell requires the action of a particular
DNase, which may be called a DNA translocase. Either magnesium or
manganous ions are essential for entry as they are for the enzyme
activity in vitro, and the in vitro products consist of oligonucleotides
similar in size to those appearing outside cells during DNA uptake.
Rudin et al. (1974) studied the factors that affect the compe-
tence for transformation in Staphylococcus aureus , using both plasmid
and chromosomal markers. The optimal pH and temperature for transforma-
tion are 6.75 to 7.0 and 30°C. Calcium ions are required for transfor-
mation. The maximal number of transformants is obtained after 20 min-
utes contact between cells and DNA.
In Bacillus subtil is 10% of the recipient cells are transformable
+ + + -f by aro trp , , M_s , and tyr determinants located on chromosomal DNA
(Bettinger and Young, 1975). However, the frequency of transformation of plasmid DNA has been reported to be several orders of magnitude lower (Erlich, 1977; Gryczan et al ., 1978).
Kono et al. (1977) introduced by transformation a tetracycline resistance plasmid and chloramphenicol resistance plasmid of s s
7
Staphylococcus aureus into jl. subtil is . Abnormal amplification of
tetracycline resistance plasmid was observed in jl. subti 1 i .
al studied the relationship between the Canosi et . (1978) molecular structure and transformation efficiency of some aureus
plasmids from Bacillus subtil is . They found that the monomers had less than one thousandth the activity of the multimeric plasmid DNA.
Gryczan and Dubnau (1978) constructed and studied the prooerties
of chimeric plasmids in Bacillus subtil is . They observed that plasmid
DNAs that were endonuclease-treated and unligated gave no detectable transformation, and as expected, that linearized plasmid DNA cannot
transform _B. subti 1 is .
Chang and Cohen (1979) reported the procedure for plasmid DNA
i polyethylene glycol (PEG) transformation in J3. subti 1 that involves induction of DNA uptake in protoplasts and subsequent regeneration of
7 the bacterial cell wall. The procedure is highly efficient (4xl0 transformants per pg of plasmid DNA) and yields up to 80% transfor- mants. Those authors reported that the frequency of transformants observed for CCC plasmid DNA occurred at a frequency of one to three orders of magnitude higher than the frequency observed for nonsuper- coiled circular plasmid DNA and linear plasmid DNA.
Contente and Dubnau (1979) studied the kinetic properties and the
effect of DNA conformation on the plasmid transformation of J3. subtilis .
They found that: (a) Competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T^ ligase restores transforming 8
activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) The DNA concentration-dependence of plasmid transformation is first order; and (g) Plasmid transformation
3 4 proceeds with a low efficiency, requiring the uptake of 10 and 10 DNA molecules per transformant. .
MATERIALS AND METHODS
Bacterial strains . The strains used and their characteri sties
are 1 i sted i n Table 1
Media . Difco Nutrient broth (NB) as well as Luria broth (LB)
(10 g Bactotryptone, 5 g yeast extract, 5 g NcCl per liter). For Luria
agar (LA,) and for Nutrient agar (NA), 20 g of agar were added to 1 liter
of LB or NB.
Minimal medium (MM) (Dobereiner and Day, 1976) contained in one
liter: 5.0 g succinic acid, 1.0 g (NH^SO^ 0.1 g NaCl , 0.2 g MgS0 *7H 0 4 2
0.1 g K H P0 , 0.4 g K H P0 , 0.2 ml (1% solution) NAMo0 ‘2H 0, 0.2 ml 2 4 2 4 4 2
(10% solution) CaCl , 0.1 ml (10% solution) FeCl^* (NH ) S0 was 2 ( 4 2 4 omitted for N-free MM). Amino acids were added to 100 yg/ml , purines
and pyrimidines to 2.0 yg/ml . For MM agar (MM-agar) 20 g of agar were
added to 1 liter of MM with (MM NA) or without (MMA) nitrogenous
supplements.
Electrophoresis buffer (E. buffer): 89 mM tris (hydroxymethyl) amino-methane (Tris), 2.5 mM di sodium ethyl enediami ne tetra acetate
(Na EDTA), and 89 mM-boric acid. 2
L ysozyme mix : Lysozyme (7500 U/ml , ribonuclease 1 (10 mg/ml), bromophenol blue (0.05%) in E buffer containing 20% Ficoll. The ribonuclease was heated (98°C for 2 min) in 0.4 M sodium acetate pH 4.0 before dilution into the lysozyme mix.
9 6532 i 1 32
10
Table 1. Bacterial strains used.
Strain Species Genetic Markers Mutagen Source Used
Sp. 7 A. brasilense Wild strain Brazil
II ll ll 1 3t Brazi
125 A II ll ll Florida 2
II R R 13t-SR str rif Florida 2 ,
13t FA-1 II trp NTG This study
II 1 3t FA- trp , met NTG From FA-1
ll 1 3t FA- trp, met, ade NTG From FA-
II 1 3t FA-4 trp, met, ade, arg NTG From FA-
13t FA- II trp, met, ade, his NTG From FA-
ll 1 3t FA- met, ade, leu NTG From FA-3
USA- 5b A. lipoferum Wild strain Washi ngton
R R R R ’ C RP E. col str , Kan amp tet This study £00" 4 , D D D
13t-RP A. brasilense Kan , amp tet This study 4 ,
. R „ R R , . R C _R E. coli str Kan amp tet This study 600 68-45 , , , 11
Antibiotics were used at concentrations of 100 yg/ml for strep- tomycin, Kanamycin and rifampicin, 10 yg/ml for tetracycline and 60 pg/ml for ampicillin.
Mutagenesis . A sample of the culture was washed in sterile dis- tilled water and incubated in water with 0.1 mg/ml N methyl -N' nitro-N' nitrosoguanidine (NTG) at 37°C for 30 minutes. The cells were again washed and resuspended in sterile distilled water. A sample of the mutagenized and of an unmutagenized culture were enumerated for survival and mutagenic control values. Another sample was grown overnight in NB.
These segregated cells were then plated on NA and colonies were replicated onto unsupplemented MM agar. Colonies unable to grow on MM agar medium were characterized on MM agar supplemented with NH^ salts or pools of amino acids, purine and pyrimidines, or casamino acids plus tryptophan, or on yeast extract.
Multiple auxotrophs were isolated by serial mutagenesis of previously isolated auxotroph.
or Construction of Azospi ri 1 1 urn strains containing RP^ Rgg _45
Log phase E. coli carrying RP^ or Rg were mixed with Azospi rill urn 8 _ 4 5
(with drug resistance markers for contraselection) and incubated in a 37°C water bath for 4-5 hours. A sample was spread on
MM agar with or without NH^ supplemented with antibiotics. The colonies growing on MM agar were selected and purified in the same medium.
Conjugation of Azospirillum in liquid medium. Azospi ri 1 1 urn strains grown to log phase in NB medium, were mixed (1 ml of proto-
trophic Azospi ri 1 1 urn with RP^ and 3 ml of auxotrophic Azospi ri 1 1 urn recipients and incubated in a 37°C water bath for 4-5 hours. Samples 12
were spread onto MM agar supplemented with contraselecting antibiotics
and required nutrient, and tested for all markers.
Mating on solid Medium . Azospiri 1 1 urn strains (prototrophs con-
taining the RP^ plasmid and auxotrophic strains) were plated together
on NA medium at a ratio of 1 to 4. After 12 hours of incubation at
37°C, the cells were washed from NA plates, diluted and plated onto
selective medium for incubation at 37°C for 2-3 days and onto NA, when
the colonies were counted and later tested on unselected markers.
Isolation of Azospirillum phage . Various soils from regions
containing Azospirillum were incubated overnight with NB culture of
A. brasi lense 13t, or a mixture of strains 13t, 84 and 125 A^. The
enrichment cultures were then shaken with chloroform, centrifuged,
filtered through a millipore and overlaid onto NA in soft agar (0.6%) O with A. brasi 1 ense indicator stains 13t, 84 or 125 A^ (10 cells per
plate). The plates were observed for plaques after 1 day at 37°C.
Isolation of plasmids by dye buoyant density centrifugation .
A modified Sharp et al. (1972) method was used to isolate plasmids.
Late log phase cells from 1,500 ml of NB were washed three times with
NET buffer (0.05 M-tris, 0.05 M-NaCl , 0.005 M-EDTA, pH 8.5) and were resuspended in 20-30 ml of SNET (NET buffer with 10% sucrose). The cells suspension was incubated at 37°C for 30 minutes with 50 mg of lysozyme and 5 mg of previously heated ribonuclease A. After addi- tion of 10-15 ml of 2% Sarkosyl solution in NET, the cells were placed at 5°C for one hour to lyse.
The pH of the DNA solution was then raised to 12.2 with slow addition of 4N NaOH and with slow stirring. At pH 12.2 most of the 13
chromosomal DNA was melted and separated, whereas the supercoiled plasmid
DNA was melted and remained entangled. The pH was then lowered to 8.5
using a 2M tris-HCl solution allowing plasmid DNA to reanneal. The sodium
concentration was adjusted to 0.3 M with the NaCl and the single strands
were removed by adsorption to nitrocellulose previously washed three
times with NET buffer. The DNA-ni trocel 1 ul ose mixture was rotated at
5°C for one hour at 33 rev /min, to remove most of the single-stranded DNA.
The DNA-nitrocellulose mixture was centrifuged at 8000 rpm for 5 minutes
and the supernatant was filtered through glass wool into cellulose nitrate
centrifuge tubes and the DNA was centrifuged for 15 hours at 15,000 rpm
onto a saturated cesium chloride shelf. The six ml above the shelf was
removed and enough to bring the density to 1.65 mg/ml (Tarrand et a!.,
1978). After the addition of ethidium bromide solution, the DNA solution was kept in the dark to minimize the breakage of the DNA strands. The
tubes were centrifuged in a Type 40 rotor at 35,000 rpm for more than
40 hours. The DNA bands were visualized with a long-wave (310 nm) ultraviolet lamp. The plasmid bands were collected and stored at 5°C in the dark. The ethidium bromide was removed into isopropanol.
Dialysis of DNA was carried out against buffer (0.02 M-tris (pH 8.0),
1 mM-EDTA (pH 8.0), 0.02 M-NaCl).
Transformation-Low pH/MnCI competence- procedure . An overnight ^
culture (Enea et al . , 1 975; Gill, Alexander, and Curtiss III, personal communication), was diluted 1:100 into NB and grown in a 37°C shaking water bath to 10® cells/ml. After being chilled on ice, 10 ml of the cells were sedimented (at 8,000 rpm for 10 min at 4°C) and gently resuspended in 10 ml of cold C-l buffer (10 mM sodium acetate, 10 mM NaCl, and , .
14
50 mM MnCK, pH 5.6). The cells were held on ice for 20 minutes, then
resedimented as above and gently resuspended in 0.2 ml of cold C-2 buffer
(10 mM sodium acetate, 75 mM CaC^, and 100 mM MnC^, pH 5.6). A volume
(0.2 ml) of these competent cells was added to 10 yl of DNA and the mix-
ture was held on ice for 30 minutes. The DNA-cell mixture was then
"heat" shocked at 30°C for 2.5 minutes. The mixture was neutralized
with 3 yl of 2M-Tris (pH 7.4), diluted and plated onto NA and selective
0 media. The colonies were counted after three days of incubation at 37 C
Sterility of the DNA, and recipient cell titers were determined on NA
while untransformed revertants were determined on selective media.
1 Agarose gel electrophoresis . The several plasmids in Azospi ri 1 urn
was resolved by agarose gel electrophoresis using a variation of the g method of Eckhardt (1978) wherein 2-3 ml of the culture (around 2 x 10
cells/ml) was centrifuged and the pellet was resuspended in 50 yl of
20% F i col 1 solution in E buffer. Into each well in the prechilled
agarose gel was added 15 yl of lysozyme-mixture (within this section)
and 10 yl of cell suspension. After 30 minutes, 30 yl of 1% sodium
dodecyl sulfate (S.D.S.) +10% Fi col 1 solution was layered onto each well followed by 50 yl layer of 1% S.D.S. + 5% Ficoll. The plasmid DNA was electrophoresed in the cold for 8 hours at 2m A and then for more
than 18 hours at 17 m A (80 V.). The gel was stained with ethidium
bromide (0.5 mg/1) of water) for more than 30 minutes and then photo-
graphed under UV light (254 nm) with Polaroid type 57 film through a red filter.
Electrophoretic elution of DNA from gel slices . DNA was sepa-
band. rated from the gel by dialysis (McDonell et al . 1977). The plasmid ,
15
visualized as described above, was cut out and placed in a dialysis bag together with a small amount of low conductivity buffer (5 mM-Tris,
2.5 mM-acetic acid). The bag was immersed between two platinum elec- trodes in buffer in a shallow plastic box, and 200 V was applied for
1 5-20 mi nutes.
Crushing DNA from gel slices . DNA was extracted from the gel crushing the gel slice with a pi pet in a small tube containing the recipient cells suspended in the cold buffer. RESULTS
Isolation of Phage
No phage were found in spite of an extensive search using soil from (a) a field in Florida where strain 125 A2 was isolated, (b) three fields in Brazil where several strains of Azospi rill urn were found.
Without phages we were unable to develop a procedure for transduction.
Conjugation
Conjugal matings between RP^ carrying donors and appropriate recipient strains of Azospi ri 11 urn were carried using a variety of methods with only a very low frequency of transconjugants (Table 2) being detected.
Frequency of transfer obtained using previously described mating pairs was very low. The presence of RP^ in the donor strain used was confirmed by gel electrophoresis.
Transformation
Using dye buoyant DNA
DNA from two steps after extraction from Azospi rill urn strains were used in early transformations. One donor DNA was from cells after lysis, melting and extraction with nitrocellulose but before centrifu- gation. This fraction will be referred to as "total plasmid" DNA. The other donor DNA was from bands separated by dye buoyant, centrifugation.
These DNAs were used to transform recipient strains to prototrophy or
16 i 3 0 0 0 0 i 0
17
Table 2. Frequency of transconjugants using different donor and recipient strains.
Strains Selective No. Cells No. Frequency Donor Recipient Markers Plated/ml Transconj. of (recipient) Transconj.
8 1 0* A Azospi ri 1 1 um Azospi ri 1 1 um trp 8xl0 O
(1 3t) 1 3t FA-1 D R R 7 8 3 Pseudomonas E. coli C Kan , tc , amp 63x1 49x1 7.7xl0" 60Q RP < 4 )
R R D 7 5 1 1 X O E. co 1 Azospirillum Kan , tc , amp 2x1 7.7x1 " " (13t) TRPT5
R 8 0~ 7 Azospi ri Hum Azospirillum tc 1 .35xl0 90 6. 6x1
1 3t-RP 1 3t FA- 0” 4 R 8 7 Kan 1.35xl0 20 1 . 5x1
1 R 8 — amp 1 .35xl0 13 OX o
8 7 ade 1 .35xl0 13 1 .OxlO" 8 -7 met 1 . 35x1 20 1 . 5x1
8 -7 trp 1 . 35x1 18 1 . 3x1
* -2 No colonies on 10 dilution. i ,
18
to resistance to antibiotics. Table 3 shows the range of frequency of
transformation obtained using 10 yl of total DNA or of DNA from either
the lower or upper bands of DNA obtained after centrifugation. The
ability to transformation to prototrophy and to antibiotic resistance
was found in "total plasmid" and in the plasmid bands (Table 3). Two
bands were always seen after centrifugation. DNA from both bands were
able to transform to prototrophy.
The recipient cells used in Table 3 were made competent by the
low pH method (Enea et al . , 1975; Gill, Alexander and Curtiss III.
personal communication). Having established that Azospi rill urn can be
genetically transformed at high frequency using cells made competent by ++ the low pH/Mn regimen, we proceeded to evaluate the factors in the
method developed for _E. col that are essential for Azospirillum . +
Age of cells . Table 4 and Figure 1 show the frequency of trp
transformants as affected by age of cells and by length of the incuba-
tion time of DNA with cells. We tested cells of 4, 5 and 6 hours after
transfer from an overnight culture. The optimum age was determined by
the time required for incubation of cells with the DNA solution. The
best results were obtained using cells of 6 hours of age. The cells
were maximally transformed after 1 hour of incubation.
Length of cold phase . Table 5 shows the frequency of ade
transformants using lower band DNA while varying the time of incubation with the DNA (cold phase). For the ade gene, the best period of time of cold phase was 30 minutes, the longest time tested. + Optimum pH . Table 6 shows the frequency of leu transformants varying the pH of the buffer. pH 5.6 seems to be the best one. ——- — — — — — —— — —— ——J — —
19
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age <4- CO on O r— ^ - of i— CO ^d" ^d LO LO LO LO LO co CO CO CO CO DNA Effect with 4-> co ro ro ro ro ro co ro ro CO ro CO CO CO CO CD c: <: 1 i 1 1 1 1 1 r— DZ 10"^ Table C S- v on O i Q OO 1 1 1 1 1 1 colonies S- CD S- Cl No 03 S- s: 4-> 21 frequency transformation time of incubation with DNA Figure 1. Effect of age of cells and incubation time with DNA on transformation frequency. J — — • " — — — —— 22 LO I 1 1 x}* co • 1 1 1 CD O 0 • 4- 1— r— , O 0 cr co O X X 1— r— qj z 1— LO LO X X • Z fO r^ 1— , Li- Z V CO • • f— CXJ . C\J CXJ CXI CXJ + • 0 0 0 0 • CO X 1 r— r— r— ade O Z O X X X X ^ ro r^. LO CXI 0 variation Z LO CXJ 0 of 1— CXJ CO 1 time — E frequency i CU T3 O CD 0 0 0 0 0 4-> 1 r— r— 1— 1— phase • ro X X X X X O r~ 1— CXI CXI CXJ CXI 2: a_ Cold transformation z c co of O 2: r— •r- Q r— 4-> CD •— (O 4- O Z _Q O •«— O LO 0 0 0 13 JZ E 1 (XJ CO Effect U CD *!” on c E 1—l *r— ^ I— z 5. o 4- co "— O 1— co 13 r— Z CD CD CO CD CD Table CDcnoCD —JZ 4-J CO CO co CO CO z 1 1 1 1 1 a) z c c < Z •1— *i Li- U- Ll. Ll_ Ll. o CL 03 •r- Z 4-> 4-> 4-> 4-> 4-> LO O 4-> CO CO CO CO CO cd CD C/0 1— 1— r— , , •r— C£ Z o Z o Z *r- o O < 03 4-> +-> 4-> 4-> 4-5 Z 21 Z CO CO CO CO CO 4-> — t 1 1— 1 O O — — ,— Q OO —— —' ——5 — — —— — 23 1 to to • 1 O 1 1 • 4- i o o o cr oo X r r— I— aj c: CO X S- H3 to oo V V u_ S- • • l r— oo 4-* o o • to r— 1— * * o c: X X o O ^ 03 LO o S- • • . 1 oo + h- buffer leu to i r— E of i— \ r*> CD “O o o o o ( C_D CD i i i +-> X X X X • 03 C\J o OJ o incubation O r— • • • • ^ Cl_ CTi cn CT> frequency 31 O to o o Q. • • • • DNA LO LO r^. 00 of c C to pH •r- Q r— 4-> (D transformation «3 4- CD C o o o o of J3 O •«- CO oo oo oo =3 -c E CD CD 4-5 cE-r; l—i *r 1 Effect on C 4- CO -— o O 1— CO 6. r— s~ to to to to CD CD JZ CDCJ ' «=C Table +-> C to to to to CD JZ i 1 •r— t— < a: 1 CD ’Ia o c o S- -r- CD O C 03 -+— 4-) 4-> 4-5 C ^ S- 00 00 OO 00 1 O Q -M 1 1 i O (/") . 24 Length of heat shock . Table 7 shows the frequency of transformants varyinq the time of heat shock. Two and one-half minutes of incubation at 37°C was the optimum length of heat shock. ++ ++ Requirement for Mn Table 8 shows that when Mn is added during the CaCl treatment of cells there is an increase in the frequency ^ of transformants DNA concentration . Table 9 and Figure 2 show the effect of DNA concentration on the frequency of transformants. Six-hour-old cells were incubated with DNA for 30 minutes. Concentrations lower than 10 pg/ml failed to transform the met gene and 30 yg/ml was in the plateau region of the response curve. Requirement for circular DNA . Transformation frequency using DNA from the lower band separated by dye buoyant density centrifugation pro- cedure was compared to the frequency using DNA passed several times through a syringe (Table 10). This shearing procedure reduced transfor- mation frequency by nearly 2 log cycles. Transformation using DNA from gel electrophoresis bands Tables 11, 12, 13, 14, 15, and 16 show the results of these experiments. In some cases, more than one marker was transformed by the same plasmid and in others, the same gene was transformed by two or more different plasmids (Figures 3 and 4). Inter and intra species transformation frequencies Transformation into the 13t auxotrophic recipient strain was carried out using DNA from other isolates of A. brasi 1 ense and from 25 Table 7. Effect of time of heat shock on transformation frequency. 10“ 3 Frequency of Transformants (x ) Time of Heat Shock (min ) Markers 0 1 2 2.5 3.0 3.5 * trp 0 0.3 2.7 2.0 1.8 0.9 0 0.5 0.8 4.1 3.1 1.0 0 0.43 0.8 3.2 2.7 1.8 Average 0 0.41 1.43 3.10 2.53 1.23 *1 3t x FA-3. 26 ++ Table 8. Effect of Mn concentration on transformation frequency. _3 Frequency of Transformants (x 10 ) MnCl Concentration Markers 2 None 50 mM 75 mM 100 mM 125 mM 150 mM leu 0.4 1.8 2.0 2.5 1.5 1.1 ** trp 0.2 0.8 1.0 1.8 1.0 0.9 ** met 0.2 0.9 0.8 1.2 0.8 0.5 ** ade 0.1 1.0 1.2 1.6 1.1 0.8 Average 0.225 1.125 1.25 1.775 1.1 0.825 *13t x 13t FA-6. ** 13t x 13t FA-3. — — — — — —- —’ 27 CO CO CNJ C\J 1 1 1 • o o o o M— LO LO LO LO 1 1 f 1 • to 1 1 1 X X X X cr 3 O o o o LO LO CO CD 03 • • • • 3 3 o o LO *3" Ll. 1— V V V V COo coo o o 00 * * * * r— r— 1 1 3 O O O O X X X X 033 LO 00 LO frequency. 10 1 r-* r^ LO LO Q • E C\J CO CO U \ on CD O 3 C\J LO CO O LO 5 * CO 3 concentration o •p- o 4-> CD •— 03 t4- o c -O O o o o o o o o o 3 _3 co co 00 co CO co CO co O CD 4-> ' c E DNA of <4- tO 4-5 O i— CO 3 i— 3 LO LO LO LO LO cnuCD CD —-3 c Effect CSJ Io CO CO CO CO CO CO CO CO 9. CD 3 I I I I I I I I 3 •T— *1 c | c c c < < o CL 03 Ll. Ll_ Li_ Ll_ Li_ Ll. •r- 3 to U 4-> 4-> 4-5 CD Table CD OO CO CO CO OO co CL 3 O o 3 CJ 3 -r- O < 03 4-J 3^3 CO OO CO CO CO CO O 4-5 Q OO 3 CD LxC3 rt3 CD 28 frequency transformation Figure 2. Effect of DNA concentration on transformation frequency. r — —— — 29 LO 1 • c 1 4- 1— o • CO >< 1 cr c CO X CD 03 c LO S- S- • u- 1— 00 # co • 4- c >> • CO 1 o CJ o c X 1 c 03 CO X CD £- o LO 13 • c r— 00 CD 4— CO i sz r- E o • r— CD TD +-> O CD 00 CO fd 4-> cz o • 03 ' g O r— o ^ Q- 4- CO c: 03 c< w S- o r- +-> •r- Q r— -M CD c 03 4- O c o o o -Q O •i— CO co Z3 jz JE C CJ CD 4-> C E *r- Q H-r- ^ h- “O— •iE CO 03 f— CL cn 4- co -—>. c= O i— co •1— r— S- CD CD S- CD CD JZ 03 cn o — CD C -C CO 4- O "O 03 CD t— +-> T3 ZJ CJ CD 03 CJ CD co CD S- 4- CD ZD JZ — 4- CO CJ L'J 4-> JZ CO co • CD £Z 1 — i O •i *i < < ' CL 03 Ll. Ll. •r- S~ CD CJ 4-> 4-> 4-> r— CD CO CO CO JD CC i— i— 03 h- oo C\J c or o: C- *r— CO co O 03 i c 2: s- 4-> +-> O O 4-> CO CO CD CO 1 r— S- CD %- 4— 03 •i S- — v — —^ — —^ 30 • o LO C\J C\J • H- X o o X o o 1 I — O 00 X X r— t 2: c X X X X X X rd O CO r— o CO o S- • • h- CD LO DNA 00 1 •— E r— CD X5 O CD 00 00 CO CO 00 CO +-> o o o o o o • rd O r— 2: a. electrodialysed c o c -r- ^ S- £- S- 4-> electrophoresis. O Q c d d :3 13 =3 with • 1 • • 03 l r— o O o CD JO Jd E E E x: -d _d E 3 4-> •r- (J -r- 1 1 o o O ,— 1— ci ^ CO CO CO genes after <4— r— d lo co LO C£) LO LO biosynthetic DIOCD CD JZ segments c of gel 4-> d CO OO 00 CO CO 00 dilution. CD d 1 1 1 1 •r— •!— < •< < < ci CL rd Ll Ll Ll Ll Ll Ll excised •r— S— CJ +J 4-> 4-> 4-> 4-> +-> 4-> Transformation CD 00 CO CO CO CO CO CO DC 10"^ T3C "O from C on ra rd jO _Q CD CD CD O * * CD CD d -r- * * * * colonies -“ S— •i— r ^ X- CD o c rd (/) CD 1— CM CD — CM V c z d d — d “s. * V X JD o o 4-> r— o o rO Q OO CD z 4-> 4-> z: 4-> 4-> h- CD CO OO CO CO No d a> S~ a) rd "O :e rd 1 " — — — — — 31 g LO LO «3- o O O 0 0 1 1 . 4- * 1— t o cn K X X X X ;z: c * LO CM LO DNA in 1— *— E 1— \ CD "O O CD CO CO CO 00 CO CO +-> O 0 0 0 0 0 1 1 1 1— r~~ • 03 1— O r- 2: Q- electrodialysed C O C 4- *r— 21 S- S- %- O +-> O C sz c: :3 ZJ 13 •1 • — •1— — 1 O electrophoresis. 03 0 0 with CD -O _C E E E JZ JZ JZ E =3 +J •r- CJ -r- O O O 1 1— 1 (- —c 2 CO CO co genes after 4- CO O 1— co r— LO co LO LO LO LO CD CD JZ biosynthetic CDO — segments of gel 4-> C CO co 00 CO CO CO 1 1 1 1 1 1 CD C dilution. •f— »r— < c Transformation cl: i r— 10 "O T3 from c C on 03 03 JD JD 12. CD S- CD _X S- 4-> CD 03 E —1 —: —— ' —— ——x^ —— 32 • 4- * LO * CM LO * O (/> * o * o O * z c * 1— * f r— * ra o X o X X o 1 LO 1— • 1 • DNA CO 1 E CD TD O CD OD CO CO 00 00 CO 4-3 o o o o o o • ra r— r— r— t— r— 1— electrodialysed O r— q_ electrophoresis. with c o < 4- -r- z: S- S- s- O 4-> o c c: C =3 Z3 =3 genes ra •1 •i •r~ o o o CD -Q -SZ E E E JZ -C _£= E 3 4-> - - •r- CJ 1 o O O 1 ( , 1— c ^ 00 OO oo after »— biosynthetic segments 4-3 c oo 00 CO CO oo OO CD I 1 1 of C 1 i 1 •1— *1 < < excised 10"^ Transformation -O -o c SZ on ra CD •K * CD * K * colonies 13. * c: N *>— X—N, S- *r- i CM i CM O c ra CD *• 1 *— CD v— O c: zr s- c: C U 4-3 4-3 O Q o 4-> o 4-3 4-> CD No Table Q OO z: CO oo z CO 00 oo * * * * * S- CD S- QJ ra q +->1 —— —— ' — — —— — — —* ——X ——" — — — — 33 CO <3* LO LD 1 i i i 1 4- o o O o o o O • CO r— i r— p— i i i cr c X X o X X X X X 1 CD 03 LO i *3- CD CXJ 1 S- S- 00 • • • Ll. 1— o • v CO CO CXJ CD LD • o O * LO CXI CO CO 4- 1 r— * o o o o o 1 1 1 1 C • CO X X * 1— CD o c: C\J * X X X X X E 03 CXJ CD * CXI LO CO LD 1 CD S- • o • • CD h- 00 CO CD CD “O CD sz CO CO • 13 r— E CD CD CD CD CD CD CD CD o CD T3 O O O O O O— O O p— p— t O CD i r— t— r— E 4-> X X X X X X X X o • 03 LD LO LD LO LO LO LO LO S- O r— ^ Cl. 4-> C • CD C c •i— *i o Q- 03 4-3 4-3 4-> 4-> 4-> 4-3 4-3 4-3 •r— •r- S- CO CO CO CO CO CO CO CO 4-> 1 1 1 CJ 4-> i r— r— r— i Z3 CD CO r— QZ •r— -a CXI i o • • • 1 “O “O • "O c c: T3 c c -X -X 03 n3 c 03 o * * •X X JD JD 03 JD •X * -X * •X •X JD to CO * X * * * * -X •X i— i i CD *— p— p— c CD x x X—>. •% — « X CD CD r— CD •r“ “O CJ r— CXJ CO 1— CXJ CO cn CD CD CD c *« -* «• *> > *' ** ** * v Q C CD •1 — — — — — CD o •r— -£Z 1 CXJ oo CXI CXI CXJ CXJ CXI CXJ 4-3 T3 JZ f— S- 03 00 CO d d Cxi d d d d d CO C "O 4-> o O S- 13 CO CO CO CO CO CO CO CO CD o S- S~ u 4-3 1 C &- 1 1 1 1 1 i i i o •r- Z3 CD O CO (_> CD 4-3 4-3 4-3 4-3 4-3 4-> 4-> 4-3 O CD JZ O o o CD CO CO CO CO CO CO CO CO CO 1 Li_ 2: v— ( p— i t i— JD r— r— i X •X •X •X X 03 X -X •X -X h- -X •X •X X •X -X CO S- CD £- 4- 03 4-> •r— :e CO ))) 43333 000 00O0 . 34 Table 15. Transformation with DNA removed from crushed gel segments excised after electrophoresis. Markers Donor Recipient No. No. Cells No. DNA Strain Repetitions Plated/ml T ransf (gel slices) )* 7 4 trp 1 3t(l 13t FA- 3 4. 25x1 1 .3xl0 ) ** 7 4 1 3t (2 13t FA-3 3 3.8 xlO 1 . 2xl0 *** 7 ***** 1 3t (3) 13t FA- 3 4. 62xl0 0 7 1 3t (4) 13t FA- 1 9.6 xlO 0 . )* met 13t(l FA- 7 4 13t 2 4 . 25x1 1 . 2x 1 ** 7 4 1 3 1 ( 2 13t FA- 2 3.6 xlO 4.7x1 7 4 1 3t (3) 1 3t FA- 2 4.5 xlO 6.0xl0 **** , 7 ***** 1 3t (4) 1 3t FA- 1 9.6 x 1 0 )* 7 4 ade 1 3 ( FA- t 1 13t 2 4.25x1 1 . 5x1 7 13t(2)** 13t FA- 2 3.6 xlO 0 kkk 7 ***** 1 3t (3) 1 3t FA- 2 4.5 xlO 0 kkkk , 4 7 1 3t (4 1 3t FA- 1 9.6 xlO 0 )* 7 arg 13t(l 13t FA-4 1 9.8 xlO 0 ** / v 7 1 3 1 ( 2 1 3t FA- 1 9.5 xlO 0 *** 7 ***** 1 3t (3) 1 3t FA-4 1 9.0 xlO 0 **** 7 4 1 3 ( 1 4 13t FA-4 1 8.5 x 1 2 . 3x1 Lowest gel band. kk Second gel band. *** Third gel band. kkkk Fourth gel band. ***** _2 No colonies on 10" dilution. 35 Figure 3 - Plasmids of 13t strain resolved by agarose gel electrophoresis. n333 00 . 36 Table 16. Transformation with heterologous DNA from crushed get segments excised after electrophoresis. Markers Donor Recipient No. No. Cells No. DNA Strai Repetitions Plated/ml Transf Strain (gel slices) ***** )* 6 trp 125 a 1 1 3t FA- 1 5xl0 0 2 ( ** / < 6 125 A 2 1 3t FA- 1 5x1 0 2 ( ) *** 6 4 125 A 1 3t FA- 2 5xl0 3 . 7x1 0 2 (3) kkkk 6 5 125 A 13t FA- 2 7.5x1 l.OxlO 2 (4) ***** )* 6 met 125 a 1 13t FA-3 1 5xl0 0 2 ( ** 6 ***** 125 A 2 13t FA- 1 5xl0 0 2 ( ) *** kkkkk , , 6 125 A 1 3t FA- 1 5xl0 0 2 (3) )**** 6 ***** 125 A 4 13t FA- 1 5x1 0 2 ( )* 6 ade 125 a 1 13t FA- 1 5x1 0 2 ( ** 6 ***** 125 A 2 13t FA-3 1 5xl0 0 2 ( ) *** , . 6 4 125 A 13t FA- 1 5xl0 1.3xl0 2 (3) 6 4 125 A 13t FA- 1 5x1 0.5xl0 2 (4) •k Lowest gel band. Second gel band. kick Third gel band. kkkk Fourth gel band. k kkkk O No colonies on 10 dilution. 37 Figure 4 - Plasmids of 125A2 strain resolved by agarose gel electrophoresis. 38 one strain of A. 1 i poferum . Table 17 shows the transformation frequency when the trp marker was selected. Similar results were obtained when met and ade were transformed (Tables 18 and 19). Frequencies of trans- formation were comparable in all cases suggesting that classical restric- tion is not active in these particular crosses between Azospirillum . — —— — —— —— — —— —— —— — 39 CO If-’ 1 1 1 i i 1 1 i 1 • t/> o o o o o o o o o 1 i ?— 1— I — 1 cr c I— 1 i O) n3 X X X X X X X X S~ S- LO co LO • • • • — r u. 1 o CO r^. • • • • o CO OJ 4-* LO LO LO C\J CO LO • (/) o o o o o o o o 1 1 1 o c: * r— i r— r— r~~ 2^ 03 o X X X X X X X X s- CO CD CO i— CD OJ o OJ cr> o homologous CO CO 0J r— OJ oo with CO 1 r- E CO co 00 CO CO o o o o o co CO CO 1 1 1 CD "O r— r o o o o 1 O CD X X X X X r r— 4-> CTi 1 CO co X X X X bands. • 1 03 r LO o co co o OJ o O r— a. C\J CD co CO LO cr> CD LO Azospirillum DNA. of centrifugation heterologous 4-> c c CO CO CO CO 00 CO co CD CO CD -r- 1 1 1 1 1 1 i 1 1 •r- ro c c =t c c and -2 17. 10 density -X -X * -X X •X -X X SC S- s- S- s- s- on •r— CD CD CD CD (D CD CD CD 03 3 CL 3 CL 3 Q. 3 CL Table S- S- o CL o CL o CL o CL O 4-> CD 1 3 1 =5 1 3 1 3 c 00 C o o 1 1 1 1 1 1 1 1 buoyant o < colonies 2: OJ OJ 4-3 4~> CD LO LO o_ CL OJ OJ 1 1 oj C\J oo oo C£ or c c Dye No 1 i OO 00 OO 00 ZD ZD CD -X oJ 03 H +->l —: — ——- ——‘- —— — — —— — — — 40 • LO I I I I I I I I • i—n <—\ (—^ t—\ t—i t—> r 4- 1 O O o o o O O O — 1 1 1 • CO CZ i i i i i cr d X X X X X X X X CD fO 1 LO LO or 03 CM d d V • 1 LO LO L - l co LO O LO LO LO CO CO CO CO 4-’ o O O o o o o O 1 1 • CO * 1 i r— r— I— I O d * X X X X X X —X X ^ <0 o LO LO co CO CM r LO d • « • • • • • 1 co CO LO CM CM co homologous CO . r— E CO CO CO co co o o o o o LO LO CD TD 1 I 1 1 1 o o o o 1 — i 1 • O CD X X X X X 1 4-> 03 CO 1 LO X X X X IS) with • 1 03 LO o CO I CO 03 CM o 00 O r— • • d ^ Cl CM i LO oo CO LO 03 fl3 JO d o • r— 4-> Azospirillum DNA. rtf 03 =3 4- +-> •r— d d CO CO CO CO CO CO CO CO CO d *. 4-> CD 1 1 i 1 1 1 1 1 i of *r~ 03 1 1 cn i 1— i i r— i I d CD •i -a dilution. 03 Transformation d with cn >> 4-> and •i 10 L0 * * * X •X •X X X d d d d d d d S- CD d on CD CD CD CD CD CD CD CD “O 18. d s CL 3 Cl 3 CL 3 CL • — 4-> 1 O CL o CL o CL o CL 03 i— 03 f— 03 1— 13 13 d d d 03 4-> o CD 1 1 1 1 1 1 1 1 >> colonies Table d go d O o o C\J CM 4-> 4-> 13 Q < Q LO LO CL CL CM CM 1 | CD CM CM CO GO CC CC d CD _X d 4-> 03 CD —o— — —— — — — — — — — 41 LO *3* *3* 1 1 1 M— l 1 1 1 1 1 • (/) o o o o o o o o o 1 1 1 1 r 1 1— f CT C f 4— LO LO LO CO CO CO • CO * o O o o o o o o 1 1 1 O C * i i 1 i r ra o X X X X X X X X l. CO CO CD 1 LO r— i— • • • • • C\J CXJ homologous with CO 1 r— E 00 00 00 co co o o o o o LO LO LO 1 1 1 urn CD TD r— r O o O o 1 CJ CD X X X X X r r i 4-> CD CO 1 LO X X X X (/> • as 1 LO o CO i co o CXJ o "O O r— • c 2: q- C\J LO 00 CO LO CD CD CD OS Azospirill DNA. JO C o •r- 4-> 03 CD of 13 heterologous M— 4-> •r— c c CO CO CO CO CO CO CO CO co CD *i 1 1 1 1 1 1 1 1 1 •t— as •O dilution. and as s- CD >, 19. 4-> m 10 * * * K * -X -X c %- S- S- S- CD S- X S- S- on Table CD CD L- CD CD CD CD CD “O s CL a) CL 3 Cl £ CL o CL s CL o CL o CL 4-> c =3 o =3 1 3 1 3 c •i i ra L- fO i 1 i | 1 l 1 1 >> colonies O S- CD o SC 4-> £Z cxj CXI 4-> +-> 13 O CO O c CXJ CXJ 1 < LO LO CL CL 1 C\J CXI CO CO DC Cd < et No Q i i CO CO CO CO ZD ZD S- CD CD rd -u z 03 , , , , DISCUSSION Azospi ri 1 1 um species have been reported to have beneficial al The mechanism of growth stim- effects on grasses (Smith et . , 1976). ulation may be through the contribution of nitrogen fixation (Day et al . 1975; Van Berkum and Solger, 1979; Neyra, 1978), phytohormone produc- tion (Tien et al., 1979), or other effects. The mechanism of stimula- tion is being studied in field plot experiments (Smith et al . 1976), in isotope dilution experiments ( De- Pol 1 i et al . 1976), and using biochemical techniques (Tien et al., 1979; Novick, 1980). Bacterial genetic approaches are also being applied to the problem (Wood, personal communication; Elmerich, personal communication). The purpose of the research described here was to contribute to the genetic studies by developing a gene transfer system for genetic analysis of Azospi rill um . Of the three modes of gene transfer found in bacteria, two (conjugation and transduction) were not of value here. The third method, transforma- tion, was shown to be a useful tool for the genetic studies. In order to develop a gene transfer system for genetic analysis of Azospi ri 11 um we needed to obtain drug resistant or biosynthetic mutants. Since mutagenesis with either nitrous acid or UV light was unsuccessful, we reluctantly turned to NTG (Adelberg et al . 1965). Mutations resistant to streptomycin, rifampicin, chloramphenicol, and spectinomyci n have been obtained (Wood, personal communication), but resistance to other antibiotics have not been found. Among several 42 , , 43 thousand survivors of NTG mutagenesis a single mutant was found requir- ing tryptophan. After subsequent sequential mutagenesis single muta- tional events occurred that led to requirements for methionine then adenine then leucine. No duplications of a requirement were found. With some other bacteria as high as 40% of the survivors of NTG exposure are auxotrophs (Adelberg et al . 1965). Haploid Aspergillus yields 10% mutants among its survivors of UV mutagenesis. Such low mutagenic frequencies are more commonly found in polyploid organisms. Elmerich (personal communication) obtained very low transfer frequencies in RP^ mediated conjugal matings of Azospirillum , but did find some linkage of transferred genes. We have also found very low gene transfer frequencies but chose not to develop this as a mode of genetic analysis. Transduction requires the availability of phages able to carry host cell DNA at a reasonable frequency. Despite extensive effort, using conventional techniques, we were unsuccessful in finding phage for Azospirillum in soil from Brazil or Florida. In both soils others had reported finding Azospi rill urn (Dobereiner and Day, 1 975; Milam, personal communication). In the absence of phage we could not develop transduc- tion as a tool for gene transfer. Bacterial transformation has been described in a variety of bacteria (Lacks et al., 1974; Cohen et al . 1972; Lederberg and Cohen, 1974; Gryczan et al., 1978; Contente and Dubnau, 1979; Gromkova and Goodgal , 1979; Sparling, 1966 and others). At about the time we were successful in obtaining high frequency transformation using a recent competency method (Enea et al., 1975; Gill, Alexander, and Curtiss III, i 44 al reported transformation personal communication), Mishra et . (1979) in Azospiri 1 1 urn at a very low level. They transformed for resistance to three antibiotics in A. brasi lense Sp 7. Carr-Dykstra (1978) examined several isolates of both species of Azospirillum and found that each contained a variety of plasmids. The total quantity of DNA contained in plasmids constituted a signif- icant proportion of the molecular weight of the chromosome of well studied bacteria such as _E, col . This, plus the observation that many large plasmids were present in all cells, suggested that the large molecules might well contain genes of considerable importance to cells of this genus. When plasmid DNA from prototrophic donors was used to transform auxotrophic recipients, a high transformation frequency was observed for these biosynthetic markers. Further, plasmid DNA from donors resistant to streptomycin and to rifampicin was able to transform these characteristics to sensitive recipients. When the plasmid DNA was further resolved into two fractions by dye buoyant density centrifugation, both bands carried the ability to transform for the markers mentioned above (Table 3). When Azospi ri 1 1 urn cells were made competent by a method developed for ji. col i (Enea et al . , 1 975; Gill, Alexander, and Curtiss III, per- sonal communication), transformation frequencies for nutritional markers were obtained that were encouragingly high. Subsequent studies were designed to evaluate the parameters necessary to develop optimal competency. One requirement for the cell to be transformed is to be in the competent stage. Ayad and Shimmin (1974) defined competence as that 45 transient physiological condition of certain bacterial cells in which they are able to take up DNA from the surrounding medium and incorpor- ate a portion of the DNA permanently into the recipient genome. So, competence is a complex condition, and has been found to be entirely different in different types of bacteria. Accordingly we determined the optimum conditions for the development of competence using: 1. State of growth cycle 2. Length of cold phase 3. Variations in pH 4. Variations in the "heat shock" 5. Requirement for divalent cations 6. Growth medium. In relation to the stage of growth cycle, we used cells of 4, 5 and 6 hours of age onto trp transformation experiments, varying the time of incubation of cells with the DNA solution. The best results were obtained using cells of 6 hours of age, incubated with the DNA solution for more than 1 hour (Table 4 and Figure 1). Younger cells required longer incubation times. The effect of varying the time of incubation of cells with the DNA solution in the cold is shown in Table 5. Five minutes of incuba- tion was enough to obtain transformants ; however, 30 minutes was much better. The pH of the buffer in which the cells were suspended was examined (Table 6). A pH of 5.6 was the optimal. Neutral or basic pHs were not effective. Thus, the pH of the buffer used to put the cells in contact with the DNA solution is very important. i 46 The competent cells were subjected to a heat pulse at 37°C for different periods of time to enable DNA uptake. We varied that period from 0 (zero) to 3.5 minutes. The highest frequency of transformants were obtained after 2.5 minutes of heat shock. The heat shock seemed to be important because no transformants were obtained without it. ++ The presence of Mn (100 mM) during the CaCl^ treatment of ++ cells resulted in higher yield of transformants . The role of Mn ++ in Ca mediated DNA uptake is unknown. Oishi and Irbe (1977) have listed the following possibilities to explain the mechanism by which CaCl treatment facilitates DNA transport through the _E. col membrane: 2 1. Calcium ions neutralize the negative charge of the cell surfaces thus removing a barrier and providing the donor DNA with easier access to cell membranes. 2. Calcium ions denature a particular type of protein in the membrane, thus affecting the penetration of DNA. 3. Calcium activates a latent enzyme which modifies the mem- brane structure to make it permeable to DNA. Gill, Alexander, and Curtiss III (personal communication) sug- gested that a major fraction of the DNA that is taken up by the cells after CaC^ treatment may not be reaching the inside of the cytoplasmic ++ membrane. The presence of Mn during exposure of cells to CaC^ may somehow facilitate somewhat larger numbers of DNA molecules reaching the cytoplasm. To determine the optimum amount of DNA needed for transfor- mation, concentration between 2.2 to 45 yg/ml were added to the recip- + ient cells. Trp frequency increased with DNA concentration up to + 30 yg/ml where this trp frequency was approximately 2xl0^/yg DNA. . 47 Shearing of CCC DNA isolated by dye buoyant density centrifu- gation reduced transformation frequency greatly--about 2 log cycles. Wood (personal communication) has extensively modified the gel electrophoresis procedure of Eckhardt (1978) into an effective method for resolving the plasmids of Azospiri 11 urn . The plasmids of Azospiri 11 urn were separated from each other, using this electrophoresis procedure and individual plasmids bands were cut out of the gel. This plasmid DNA was separated from the agarose by either of two methods and used for transformation. These isolated plasmid DNA fractions were effective in transforming several auxotrophs to prototrophy (Tables 11, 12, 13, 14, 15, and 16). The argument that these bands could contain fragments of chromosomal DNA that somehow spread throughout the agarose gel was considered. Accordingly segments of the gel consisting of inter band agarose was treated as were the segments containing the visible bands. Such bands did not transform auxotrophic recipients to prototrophy. Similar attempts were made using material extracted as above from agarose cut from the region of what is conventionally referred to as DNA fragments — the region of a broad band of ethidium bromide stain- ing material near the bottom of the gel. No transformants were found usi ng this material These results do not support the argument that the transformation attributed to plasmid band DNA actually results from chromosomal DNA present in the gel. It might be argued that the concentration of chromosomal DNA throughout the gel is too low for effective transfor- mation but because of some kind of DNA-DNA association that chromosomal 48 DNA migrates at high concentration with plasmid bands. This possibility is not supported by the finding that many gel segments containing plasmid DNA (Figures 3 and 4) failed to transform for the marker selected. Transformation into the 13t auxotrophic recipient strain was carried out using DNA from other isolates of A. brasi Tense and from + one strain of A. lipoferum . Table 17 shows the trp frequency when the trp marker was selected. Similar results were obtained when met and ade were transformed. Frequencies of transformation were comparable in all cases suggesting that classical restriction is not active in these particular crosses between Azospirillum species. The transformations described seem likely to be the result of recombination events. The mutations in the recipients are NTG induced rather than curing events. Cured strains with auxotrophic properties are unavailable for studies on "transformation by addition." The donor DNA isolated by dye buoyant density centrifugation and used in transformations consists of a mixture of CCC, open circles, linear molecules and fragments (Carr-Dykstra , 1978). The state of DNA in agarose gel bands is uncertain. Because it migrates in discrete bands it seems likely that it mi orates as CCC molecules. Upon staining with Et Br and exposure to 254nmUV light (for the purpose of visual- ization) many of the molecules are likely to be nicked. Previously, bacteria have been found to carry biosynthetic genes on a chromosome while unessential properties (sex, resistance, toxins, bacterioci ns , surface antigens) are carried on plasmids. Azospiri 1 1 urn appears to be a genus that carries essential genes on several linkage groups and exhibits redundancy of some genes on several plasmids. 49 From all the data that we obtained concerning the mechanism of transformation in Azospirillum we can summarize and enumerate the prop- erties of the transforming DNA that support the hypothesis that the transformations carried out in these experiments are, in fact, with plasmid DNA. Firstly, by electron microscopy observations Carr-Dykstra (1978) observed three types of molecules from both plasmid bands isolated by the Sharp et al. (1972) technique: mostly covalently closed circles Secondly, by agarose (CCC) , open circles and a few linear fragments. gel el ectrophoresi s technique, we could uniformly observe the same pat- tern of bands for each Azospirillum strain, indicating that those bands correspond to plasmid DNA rather than chromosomal fragments. 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A Taxonomic Study of the Spirillum lipoferum group, with Descriptions of a New Genus, Azospi ri 1 1 urn gen. nov. and two Species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can. J. Microbiol. 24: 967-980. Tien, T.M. , M.H. Gaskins, and D.H. Hubbell. 1979. Plant Growth Sub- stances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.) Applied and Envir. Microbiol. 31: 1016-1024. Tomasz, A. 1966. Model for the Mechanism Controlling the Expression of Competent State in Pneumococcus Cultures. J. Bacteriol. 31: 1050-1061 Umal i-Garcia , M. , D.H. Hubbell, M.H. Gaskins, and F.B. Dazzo. 1980. Association of Azospirillum with Grass Roots. Appl. and Env. Microbiol. 39: 219-226. Van Berkum, P., and C. Sloger. 1979. Immediate Acetylene Reduction by Excised Grass Roots not Previously Preincubated at Low Oxygen Tensions. Plant Physiol. 64: 739-745. Van Berkum, P., and C. B. Bohlool. 1980. Evaluation of Nitrogen Fixation by Bacteria in Association with Roots of Tropical Grasses. Microbiol. Rev. 44: 491-517. Von BLilow, J.F.W., and J. Dobereiner. 1 975. Potential for Nitrogen Fixation in Maize Genotypes in Brazil. Proc. Natl. Acad. Sci (USA) 72: 2389-2393. BIOGRAPHICAL SKETCH Elza (Rodrigues) Machado Menezes was born on April 18, 1944, in Piracicaba, Sao Paulo, Brazil. She is the fourth child of nine children of Maria da Conceiqao Bueno Machado and the late Elias Rodrigues Machado. Following graduation from Col£gio Estadual "Presidente Roosevelt" (High School) in Sao Paulo, she attended the Universidade de Sao Paulo and received the Bachelor of Science degree with a major in biological science in December, 1970. She attended the Escola Superior de Agricultura "Luiz de Queiroz" (USP) and received the degree of Master of Science with a major in genetics in December, 1974. In June, 1974, she became a faculty member at the Universidade Federal Rural do Rio de Janeiro, Department of Genetics. In June, 1976, she came to the United States and took a course in the English language at Michigan State University, East Lansing, Michigan. In January, 1977, she began the studies for her Ph.D. degree at the University of Florida, Gainesville, Florida. She is currently a candidate for the degree of Doctor of Philosophy in the Department of Microbiology and Cell Science. She married Euripedes B. Menezes in December, 1971. They have two daughters, Caroline and Jacqueline. 54 - I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. / r i iC I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Phi 11 ip t1. Achey Professor of Microbiology and Cell Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March 1981 Dean for Graduate Studies and Research