- Email: [email protected]
A novel method for the isolation of mycobacterial DNA
ELSEVIER
FEMS Microbiology
Letters I35 (1996) 7 l-77
A novel method for the isolation of mycobacterial Jorge A. Gonzalez-y-Merchand
Received 28 August
I, Iris
Estrada-Garcia Robert A. Cox *
1995: revised 25 October
DNA
‘, M. Joseph Colston,
1995: accepted 27 October
1995
Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to -7O”C, then incubated at 65°C for IO min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (I X IO” or more cells) including Myobacterium neoaurum, M. fortuitum, M. phlei and M. smegmatis. Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis. Kqwonls:
Mycobacteria:
DNA isolation; rRNA operon
1. Introduction The increasing importance of studies of amplified regions of the genome of bacterial pathogens such as Mycobacterium tuberculosis and M. leprue highlights the need for rapid and simple methods for the isolation of mycobacterial genomic DNA. Mycobacteria have a waxy lipid envelope which covers a cell wall that is typical of Gram-positive bacteria. Mycolic acids are components of this envelope [l]. Mycobacteria are difficult to lyse, for example by the
_ Corresponding author. Tel.: +44 (181) 959 3666 ext. 2347: Fax: + 44 ( 18 I ) 9 I3 8528: E-mail: [email protected]. ’ Present address: Escuela National de Ciencias Biologicas, IPN. Ap. Postal 4-870, Mexico, D.F. 06400, Mexico. ’ Present address: Escuela National de Ciencias Biologicas. IPN, Ap. Postal 4.870, Mexico. D.F. 06400, Mexico. 0378- lO97/96/$I2.00 0 1996 Federation SSDI 0378- 1097(95)00432-7
of European
Microbiological
widely used Marmur [2] method, because of the complex nature of their outer coat. As a consequence enzymic methods have been sought to weaken the cell wall and mechanical procedures have been used to rupture the cells (see Table I). We now describe a method which produces 15 pg DNA per I X 10”’ cells: pretreatment of the cells is kept to a single cooling step and mechanical disruption is avoided. The novel step is incubation of mycobacteria in 6 M guanidinium chloride at 65°C for 10 min. The method was applied to several species of mycobacteria; namely, M. neoaururn, M. ~fortuitum, M. phlei and M. smegmatis which are representatives of a wide range of fast growers, as judged by their phylogenetic relationships 131. The isolated DNAs were shown to be cleaved by restriction endonucleases and to be effective substrates in the polymerase chain reaction (PCR). Societies.
All rights reserved
a I X 10”’ cells is equivalent
’ )/
’ )/lysozyme
Proteinase K (200 pg ml lysozyme
Lipase (2 mg ml (2mgml-‘)
None
to approx.
(I) Incubation with lysozyme (500 @g ml- ‘) for 1 h at at 60°C. (2) Incubation with pronase (IOO~gml-‘)and I%(w/v)SDS overnight at 37°C. None
SDS for I5 min at 70°C
Mechanical (glass beads, 2 min, 4°C) Solvent: 0. I M NaC1/0.03 M Tris/6 mM EDTA Mechanical (French pressure cell at - 10°C). Solvent: 6 M guanidinium chloride Hypertonic shock (6 M guanidinium chloride) for 16 hat - ISOC Mechanical (glass beads, vortex mixed) Solvent: 4 M guanidinium thiocyanate at room temp.
3% (w/v)
10 mg cells (wet weight).
Reflux with chloroform/methanol (2: 1, v/v) for 6 h at approx. 60°C. (1) Heat shock (196”C/ + 100°C) (2) Incubation with 3% (w/v) SDS for 1 h at 65°C.
of cell extract
isoquick R isolation kit
15 @g
Not quoted
[I II
[lOI
[91
1l-16 Kg
Precipitation of nucleic acids with ethanol etc.
PI
Not quoted
Treatment with chloroform/ phenol etc.
with chloroform.
[71
Not quoted
Treatment
k31
[41
Reference
2.5 CLg
per 10”’ cells “1
[51
Not quoted
Yield of DNA (~g
I.7 IJ,g
’ ) chloroform,
with phenol etc. Pronase/RNAase/phenoI extraction, etc. Resuspended cells shaken with chloroform/isoamyhc alcohol/phenol (24:1:25) for 15 min. etc. 1 M NaCIO, treatment with chloroform/isoamyl alcohol, etc
Extraction
(2 mg ml-’ ) for 2 h at 37°C. K ml-
Hypotonic shock (TE buffer containing 100 pg proteinase Hypotonic shock (TE buffer/O.S%SDS, w/v) Heat (microwave oven) treatment of pelleted ceils
Lysozyme
Further treatments
Method of cell lysis/rupture
Pretreatment
of cells to weaken the cell wall
of methods used for the isolation of DNA from mycobacteria
Table I Comparison
J.A. Gonzalez-y-Merchand
et al. / FEMS Microbiology
Letters I35 f 1996) 71- 77
73
2. Materials and methods
2.2. Bacterial
2.1. Materials
M. neoaurum NCTC 108 18, M. fortuitum NCTC 10394, M. phlei NCTC 815 1, M. smegmatis NCTC 8159 and M. smegmatis mc’ 155 (a gift from W. Jacobs, Albert Einstein College of Medicine, New York, USA) were maintained on Lowenstein Jensen slopes and grown for use in Lemco broth containing 0.1% (v/v) Tween 80. In all cases, cells were in the late exponential to early stationary phase of growth at the time of harvest.
Restriction endonuclease PstI was from Boehringer Mannheim and [ (Y-~~P]dCTP was from Amersham. AmpliTaq DNA polymerase was supplied by Perkin Elmer Cents. The oligolabelling kit was purchased from Pharmacia. Oligonucleotide primers were prepared as previously described [ 121. All other chemicals were of analytical reagent grade.
strains and media
(A) Protocol for DNA isolation 1) Collect cells from growth medium by centrifugation (microfuge) and resuspend in lysis buffer (10 ~1 of buffer mg.’ of cells [wet weight]). 2) Freeze the cell suspension (ethanol/solid CO, freezing mixture). 3) Transfer to water bath and incubate at 65°C for 10 min. 4) Repeat steps 2 & 3. 5) Chill on ice for 5 min. 6) Add two volumes of chloroform reagent and mix (vortex mixer for 1 second at maximum speed).
Keep the aqueous phase and repeat the procedure once more.
7) Precipitate DNA from the aqueous phase by the gradual addition of two volumes of ethanol. Mix by inverting the tube. 8) Recover the DNA precipitate by centrifuging (microfuge); dissolve in TE buffer and keep at 4°C.
(B) Reagents a) Lysis buffer:
b) TE buffer:
c) Chloroform reagent:
6M guanidinium chloride
10 mM TrisiImM EDTAIHCI,
chloroform (24 vols.)/
ImM 2-mercaptoethanol
pH 7.5
isoamyl alcohol (I vol.)
10 mM EDTA 0.1% Tween 80 Fig. I. Protocol for DNA isolation
2.3. Agarose
gel electrophoresis
Mycobacterial genomic DNA (2-4 pg) isolated as shown in Fig. 1 was analysed by electrophoresis on 0.8% (w/v) agarose gel using standard conditions. The ethidium bromide stained gels were photographed under UV light. Lambda DNA/Hind111 fragments were used as molecular mass markers. 2.4. Restriction of DNA and Southern transfer DNA (l-3 pg) was digested with PstI according to the manufacturer’s (Boehinger Mannheim, UK) instructions. After separation by electrophoresis, the DNA fragments were transferred from agarose gels to nylon membrane by standard methods.
the primer combination of JG7 (5’-CTGCAGCCGATGGCTATCGCTTTG-3’) and RACS. The target for primer JY 13 is the sequence complementary to the Box B element of the mycobacterial ~rri A operon [9,12]. The target for primer RAC8 is positions 339-357 of the 16s rRNA coding region [ 141. The target for primer JG7 is the sequence complementary to the first 24 nucleotides downstream from the 5’-end of the PstI fragment of A4. tuberculosis (positions l-24 of the sequence deposited with the EMBL Data Bank, accession number X87944; see also [ 141). A 100 bp ladder (Life Technologies) was used as molecular mass markers. All primers were synthesized as previously described [ 121.
3. Results and discussion 2.5. Preparation
of radioactive probe and hybridiza-
tion conditions
3. I. The isolation of DNA
A DNA fragment (100 ng) containing the S-end (positions l-355) of the 16s rRNA gene sequence of M. smegmatis was labelled as specified in the manual of the oligolabelling kit, using 50 @i of [cr32P]dCTP. The baked membrane was prehybridized for 1 h and then hybridized overnight at 65°C in 0.5 M sodium phosphate (pH 7.2)/7% (w/v) SDS [13]. After hybridization, the membrane was washed under high stringency conditions: 3 X SSC/O.l% (w/v) SDS, for 20 min at 65°C; 1 X SSC/O.l% SDS (w/v) for 20 min at 65°C; 0.1 X SSC/O.l% (w/v> SDS for 20 min at 65°C and 0.1 X SSC for 20 min at room temperature.
The method presented in Fig. 1 is based on the use of the chaotropic agent guanidinium chloride for isolation of DNA [ 151. This reagent was first used for the isolation of mycobacterial DNA in conjunction with the French press [9]. Later it was noted that mycobacteria pretreated with lipase and lysozyme are susceptible to lysis by guanidinium chloride at - 15°C [lo]. The current method (see Fig. 1) is both simpler and quicker than previous procedures. Mycobacteria are resuspended in lysis buffer, frozen in a solid CO,/ethanol freezing mixture (approx. - 70°C) then heated to 65°C for 10 min. This treatment causes either cell lysis or renders cells susceptible to lysis on treatment with chloroform. After the heat treatment, RNA sediments with the cell debris so that no further steps are needed to remove it. Double stranded DNA is stable in lysis buffer (T,,, approx 75°C); as shown previously for rat liver DNA in 4 M guanidinium chloride [16]. We used guanidinium chloride which can be cooled to below - 10°C. However, guanidinium isothiocyanate is expected to be equally effective. Also, we recovered DNA from lysis buffer, after deproteinization, by precipitation by ethanol. Other methods such as the IsoquickR isolation kit (see Table I) or the use of silica or diatoms [ 171 were not explored. The procedure was applied to several species of mycobacteria (see Fig. 2a). DNA was recovered
2.6. DNA polymerase
chain reaction (PCR)
Bacterial DNA (l-100 ng) was subjected to PCR as described previously [ 121. One gene fragment coding for part of pre-rRNA extending from the leader region into the 16s rRNA gene was synthesized using the primer combination of JY 13 (5’~GGGTGGCCCCGAAGCG-3’) and RAC8 (5’-CACTGGTGCCTCCCGTAGG-3’). Another fragment coding for 342 bp of the enzyme UDP-N-acetylglucosamine 1-carboxyvinyl transferase (EC 2.5.1.7) the promoter region of the rrn A operons, the 5’-ends of the leader regions of their pre-rRNA and the 5’-ends of the 16s rRNA gene was synthesized using
5..4. Gon,-ale:-y-Merchand
rt 01. / FEMS Microbiology
Letters
135 (19%)
15
71-77
kb
23.12.3 ---c
Fig. 2. Molecular mass distribution of DNA. DNA isolated by the method described in Fig. 1 was analysed by 0.8% (w/v) agaroae gel electrophoresis. as described in Materials and methods. (a) Isolation of DNA from several mycobacterial species. Lane I, mole@ular mass markers; lane 2, M. neoaurum DNA; lane 3, M. fortuirum DNA; lane 4, M. phlei DNA; lane 5, M. snqmaris NCTC 8 159 DNA; lane 6, M. smegmaris mc’155 DNA. (b) Isolation of DNA from 1 mg to 30 mg (wet weight) of M. smegmatis (NCTC 8159). Lane I, molecular mass markers: lane 2, DNA isolated from 1 mg of cells; lane 3, DNA isolated from 2 mg of cells; lane.4, DNA isolated from 3 mg of cells; lane 5, DNA isolated from IO mg of cells: lane 6, DNA isolated from 30 mg of cells. In each case 5 ~1 of a total volume of 20 /*I was loaded onto the gel
from approx 1 X 109(1 mg wet weight of cells) to 3 X lO”‘(30 mg wet weight of cells) cells (see Fig. 2b). The product obtained was approx. 23 kbp in size and an apparently higher molecular mass fraction was always present (see Fig. 2a). The yield of purified DNA (approx. 2.5%) is similar to that obtained previously [9,11]. The DNA samples were found to be essentially free from DNAase activity because there was no change in the electrophoretic pattern (cf Fig. 2a) after incubation in the buffer used for the restriction endonuclease &I, at 37°C for 16 h. Also, as shown below, the DNAs were suitable substrates for restriction endonucleases and for amplification by PCR. 3.2. Restriction fragments
qf rRNA
(t-m) operons
Genomic DNAs were isolated as described in Fig. 1 and the number of rrn operons per genome was determined by revealing the number of PstI fragments having 16s rRNA gene sequences as described in Fig. 3. The results show that M. neoaurum has two rrn operons per genome, like M. ,fortuitum, M. phlei and M. smegmatis; whereas M. tuberculosis has a single rrn operon [ 18,191. In each case a PstI fragment of 1.2-1.4 kbp was identified.
3.3. Amplification
by PCR
fragments of M. smegmatis and M. of 1373 bp and 1267 bp respectively provide a frame of reference for the study of the rrn The
PsrI
tuberculosis
ABCDEFGHI
JKLM
kb -23.1 -3.2
^,
_ --
- ._ ” _.___..
,”
Fig. 3. Investigation of the number of rm operons per genome. Genomic DNA was restricted with PsrI, the fragments were separated, transferred to a nylon membrane and prubed with a “P-1abelled 16s rRNA gene sequence (positions I-3551 as described in Materials and methods. Lane A, M. neoclwmm DNA; lane B, sample restricted with &I. Lane C. M. ,forluitum DNA; lane D. sample restricted with /%I. Lane E, M. phki DNA; lane F, sample restricted with PstI. Lane G, M. srneKnzuris (NCTC 8159) DNA; lane H, sample restricted with &I. Lane I, M. .smegmatis (mc’l55) DNA; lane J, sample restricted with &I. Lanes K and L, no DNA. Lane M, M. tuberculosis DNA restricted with WI.
J.A. Gon,-alrr-~-Mer~hund et ~11. / FEMS Microbiology Letters 135 (IYY6) 71-77
76
Py
100 basepairs
I
JY13-
approx 1025 bp
4
)f-
approx 296 bp ---_)
approx 1320 bp
_)
Fig. 4. Region
of an operon of the rrn A family upstream from the 16s rRNA gene. The location and size of the shown. JG7 + etc., binding sites for the primers used in PCR amplification: the sizes of the products obtained primers are indicated.
operons of other mycobacterial species because their nucleotide sequences have been established. The EMBL accession numbers for the nucleotide sequence data are X87944 (M. tuberculosis, see also [14]) and X87943 (M. srnegmatis). The two PstI fragments are homologous: each fragment has the 3’-terminal 114 codons of an open reading frame which is significantly similar to a gene for UDP-Nacetylglucosamine 1-carboxyvinyl transferase (EC 2.5.1.7) at its S-end and each extends downstream to include the first approx 655 bp of the 16s rRNA gene (see Fig. 4). The binding sites for primer JG7 kb
PstI fragment studied is for a particular
pair of
are located at the 5’-ends of the above mentioned PstI fragments and the binding sites for primer RAC8 lie within the 16s rRNA gene approx. 296 bp upstream from the 3’-PsrI site. Thus primers JG7/RAC8 should amplify homologues of the 1,373 bp PstI fragment of the rrn A, operon of M. smegmatis less 296 bp of the 16s rRNA gene [ 121. The results presented in Fig. 5a reveal that a homologue of the M. smegmatis PstI fragment, is present in each of the genomes studied; the sizes of the fragments lie in the range 0.95- 1.1 kbp. The rrn A, operon of il4. smegmatis and the rrn
123456789
f&l23456789 2.07
2.07 1.5 ---_,
0.6
0.6 -
(4
lb)
Fig. 5. Amplification by PCR of regions of the rrn operons of mycobacteria. (a) Products obtained by amplifying the region from the S-&I site to position approx. 355 of the 16s rRNA gene. Lane 1, molecular mass markers; Lanes 2 and 8, no DNA; Lane 3, M. ne~aurum DNA; Lane 4, M. forruirum DNA; Lane 5, M. phlei DNA; Lane 6, M. smegmatis (NCTC 8159) DNA; Lane 7, M. smegmtis (mc’ 155) DNA; Lane 9, M. tuberculosis DNA. The faster moving bands in lane 3 are an artefact unrelated to rrn operons. (b) Products obtained by amplifying the region from approx 200 bp upstream from the 5’.end of the 16s rRNA coding region to position approx. 355 of the 16s rRNA gene. Lane 1, molecular mass markers; Lanes 2 and 3, no DNA: Lane 4, M. neoaurum DNA; Lane 5, M. fortuitum DNA; Lane 6, M. phlei DNA; Lane 7, M. smrgmatis (NCTC 8159) DNA; Lane 8, M. smqmaris (mc’ 155) DNA; Lane 9, M. tuberculosis DNA.
FEMS Microbiology
Letters I35 (1996) 7 l-77
A novel method for the isolation of mycobacterial Jorge A. Gonzalez-y-Merchand
Received 28 August
I, Iris
Estrada-Garcia Robert A. Cox *
1995: revised 25 October
DNA
‘, M. Joseph Colston,
1995: accepted 27 October
1995
Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to -7O”C, then incubated at 65°C for IO min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (I X IO” or more cells) including Myobacterium neoaurum, M. fortuitum, M. phlei and M. smegmatis. Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis. Kqwonls:
Mycobacteria:
DNA isolation; rRNA operon
1. Introduction The increasing importance of studies of amplified regions of the genome of bacterial pathogens such as Mycobacterium tuberculosis and M. leprue highlights the need for rapid and simple methods for the isolation of mycobacterial genomic DNA. Mycobacteria have a waxy lipid envelope which covers a cell wall that is typical of Gram-positive bacteria. Mycolic acids are components of this envelope [l]. Mycobacteria are difficult to lyse, for example by the
_ Corresponding author. Tel.: +44 (181) 959 3666 ext. 2347: Fax: + 44 ( 18 I ) 9 I3 8528: E-mail: [email protected]. ’ Present address: Escuela National de Ciencias Biologicas, IPN. Ap. Postal 4-870, Mexico, D.F. 06400, Mexico. ’ Present address: Escuela National de Ciencias Biologicas. IPN, Ap. Postal 4.870, Mexico. D.F. 06400, Mexico. 0378- lO97/96/$I2.00 0 1996 Federation SSDI 0378- 1097(95)00432-7
of European
Microbiological
widely used Marmur [2] method, because of the complex nature of their outer coat. As a consequence enzymic methods have been sought to weaken the cell wall and mechanical procedures have been used to rupture the cells (see Table I). We now describe a method which produces 15 pg DNA per I X 10”’ cells: pretreatment of the cells is kept to a single cooling step and mechanical disruption is avoided. The novel step is incubation of mycobacteria in 6 M guanidinium chloride at 65°C for 10 min. The method was applied to several species of mycobacteria; namely, M. neoaururn, M. ~fortuitum, M. phlei and M. smegmatis which are representatives of a wide range of fast growers, as judged by their phylogenetic relationships 131. The isolated DNAs were shown to be cleaved by restriction endonucleases and to be effective substrates in the polymerase chain reaction (PCR). Societies.
All rights reserved
a I X 10”’ cells is equivalent
’ )/
’ )/lysozyme
Proteinase K (200 pg ml lysozyme
Lipase (2 mg ml (2mgml-‘)
None
to approx.
(I) Incubation with lysozyme (500 @g ml- ‘) for 1 h at at 60°C. (2) Incubation with pronase (IOO~gml-‘)and I%(w/v)SDS overnight at 37°C. None
SDS for I5 min at 70°C
Mechanical (glass beads, 2 min, 4°C) Solvent: 0. I M NaC1/0.03 M Tris/6 mM EDTA Mechanical (French pressure cell at - 10°C). Solvent: 6 M guanidinium chloride Hypertonic shock (6 M guanidinium chloride) for 16 hat - ISOC Mechanical (glass beads, vortex mixed) Solvent: 4 M guanidinium thiocyanate at room temp.
3% (w/v)
10 mg cells (wet weight).
Reflux with chloroform/methanol (2: 1, v/v) for 6 h at approx. 60°C. (1) Heat shock (196”C/ + 100°C) (2) Incubation with 3% (w/v) SDS for 1 h at 65°C.
of cell extract
isoquick R isolation kit
15 @g
Not quoted
[I II
[lOI
[91
1l-16 Kg
Precipitation of nucleic acids with ethanol etc.
PI
Not quoted
Treatment with chloroform/ phenol etc.
with chloroform.
[71
Not quoted
Treatment
k31
[41
Reference
2.5 CLg
per 10”’ cells “1
[51
Not quoted
Yield of DNA (~g
I.7 IJ,g
’ ) chloroform,
with phenol etc. Pronase/RNAase/phenoI extraction, etc. Resuspended cells shaken with chloroform/isoamyhc alcohol/phenol (24:1:25) for 15 min. etc. 1 M NaCIO, treatment with chloroform/isoamyl alcohol, etc
Extraction
(2 mg ml-’ ) for 2 h at 37°C. K ml-
Hypotonic shock (TE buffer containing 100 pg proteinase Hypotonic shock (TE buffer/O.S%SDS, w/v) Heat (microwave oven) treatment of pelleted ceils
Lysozyme
Further treatments
Method of cell lysis/rupture
Pretreatment
of cells to weaken the cell wall
of methods used for the isolation of DNA from mycobacteria
Table I Comparison
J.A. Gonzalez-y-Merchand
et al. / FEMS Microbiology
Letters I35 f 1996) 71- 77
73
2. Materials and methods
2.2. Bacterial
2.1. Materials
M. neoaurum NCTC 108 18, M. fortuitum NCTC 10394, M. phlei NCTC 815 1, M. smegmatis NCTC 8159 and M. smegmatis mc’ 155 (a gift from W. Jacobs, Albert Einstein College of Medicine, New York, USA) were maintained on Lowenstein Jensen slopes and grown for use in Lemco broth containing 0.1% (v/v) Tween 80. In all cases, cells were in the late exponential to early stationary phase of growth at the time of harvest.
Restriction endonuclease PstI was from Boehringer Mannheim and [ (Y-~~P]dCTP was from Amersham. AmpliTaq DNA polymerase was supplied by Perkin Elmer Cents. The oligolabelling kit was purchased from Pharmacia. Oligonucleotide primers were prepared as previously described [ 121. All other chemicals were of analytical reagent grade.
strains and media
(A) Protocol for DNA isolation 1) Collect cells from growth medium by centrifugation (microfuge) and resuspend in lysis buffer (10 ~1 of buffer mg.’ of cells [wet weight]). 2) Freeze the cell suspension (ethanol/solid CO, freezing mixture). 3) Transfer to water bath and incubate at 65°C for 10 min. 4) Repeat steps 2 & 3. 5) Chill on ice for 5 min. 6) Add two volumes of chloroform reagent and mix (vortex mixer for 1 second at maximum speed).
Keep the aqueous phase and repeat the procedure once more.
7) Precipitate DNA from the aqueous phase by the gradual addition of two volumes of ethanol. Mix by inverting the tube. 8) Recover the DNA precipitate by centrifuging (microfuge); dissolve in TE buffer and keep at 4°C.
(B) Reagents a) Lysis buffer:
b) TE buffer:
c) Chloroform reagent:
6M guanidinium chloride
10 mM TrisiImM EDTAIHCI,
chloroform (24 vols.)/
ImM 2-mercaptoethanol
pH 7.5
isoamyl alcohol (I vol.)
10 mM EDTA 0.1% Tween 80 Fig. I. Protocol for DNA isolation
2.3. Agarose
gel electrophoresis
Mycobacterial genomic DNA (2-4 pg) isolated as shown in Fig. 1 was analysed by electrophoresis on 0.8% (w/v) agarose gel using standard conditions. The ethidium bromide stained gels were photographed under UV light. Lambda DNA/Hind111 fragments were used as molecular mass markers. 2.4. Restriction of DNA and Southern transfer DNA (l-3 pg) was digested with PstI according to the manufacturer’s (Boehinger Mannheim, UK) instructions. After separation by electrophoresis, the DNA fragments were transferred from agarose gels to nylon membrane by standard methods.
the primer combination of JG7 (5’-CTGCAGCCGATGGCTATCGCTTTG-3’) and RACS. The target for primer JY 13 is the sequence complementary to the Box B element of the mycobacterial ~rri A operon [9,12]. The target for primer RAC8 is positions 339-357 of the 16s rRNA coding region [ 141. The target for primer JG7 is the sequence complementary to the first 24 nucleotides downstream from the 5’-end of the PstI fragment of A4. tuberculosis (positions l-24 of the sequence deposited with the EMBL Data Bank, accession number X87944; see also [ 141). A 100 bp ladder (Life Technologies) was used as molecular mass markers. All primers were synthesized as previously described [ 121.
3. Results and discussion 2.5. Preparation
of radioactive probe and hybridiza-
tion conditions
3. I. The isolation of DNA
A DNA fragment (100 ng) containing the S-end (positions l-355) of the 16s rRNA gene sequence of M. smegmatis was labelled as specified in the manual of the oligolabelling kit, using 50 @i of [cr32P]dCTP. The baked membrane was prehybridized for 1 h and then hybridized overnight at 65°C in 0.5 M sodium phosphate (pH 7.2)/7% (w/v) SDS [13]. After hybridization, the membrane was washed under high stringency conditions: 3 X SSC/O.l% (w/v) SDS, for 20 min at 65°C; 1 X SSC/O.l% SDS (w/v) for 20 min at 65°C; 0.1 X SSC/O.l% (w/v> SDS for 20 min at 65°C and 0.1 X SSC for 20 min at room temperature.
The method presented in Fig. 1 is based on the use of the chaotropic agent guanidinium chloride for isolation of DNA [ 151. This reagent was first used for the isolation of mycobacterial DNA in conjunction with the French press [9]. Later it was noted that mycobacteria pretreated with lipase and lysozyme are susceptible to lysis by guanidinium chloride at - 15°C [lo]. The current method (see Fig. 1) is both simpler and quicker than previous procedures. Mycobacteria are resuspended in lysis buffer, frozen in a solid CO,/ethanol freezing mixture (approx. - 70°C) then heated to 65°C for 10 min. This treatment causes either cell lysis or renders cells susceptible to lysis on treatment with chloroform. After the heat treatment, RNA sediments with the cell debris so that no further steps are needed to remove it. Double stranded DNA is stable in lysis buffer (T,,, approx 75°C); as shown previously for rat liver DNA in 4 M guanidinium chloride [16]. We used guanidinium chloride which can be cooled to below - 10°C. However, guanidinium isothiocyanate is expected to be equally effective. Also, we recovered DNA from lysis buffer, after deproteinization, by precipitation by ethanol. Other methods such as the IsoquickR isolation kit (see Table I) or the use of silica or diatoms [ 171 were not explored. The procedure was applied to several species of mycobacteria (see Fig. 2a). DNA was recovered
2.6. DNA polymerase
chain reaction (PCR)
Bacterial DNA (l-100 ng) was subjected to PCR as described previously [ 121. One gene fragment coding for part of pre-rRNA extending from the leader region into the 16s rRNA gene was synthesized using the primer combination of JY 13 (5’~GGGTGGCCCCGAAGCG-3’) and RAC8 (5’-CACTGGTGCCTCCCGTAGG-3’). Another fragment coding for 342 bp of the enzyme UDP-N-acetylglucosamine 1-carboxyvinyl transferase (EC 2.5.1.7) the promoter region of the rrn A operons, the 5’-ends of the leader regions of their pre-rRNA and the 5’-ends of the 16s rRNA gene was synthesized using
5..4. Gon,-ale:-y-Merchand
rt 01. / FEMS Microbiology
Letters
135 (19%)
15
71-77
kb
23.12.3 ---c
Fig. 2. Molecular mass distribution of DNA. DNA isolated by the method described in Fig. 1 was analysed by 0.8% (w/v) agaroae gel electrophoresis. as described in Materials and methods. (a) Isolation of DNA from several mycobacterial species. Lane I, mole@ular mass markers; lane 2, M. neoaurum DNA; lane 3, M. fortuirum DNA; lane 4, M. phlei DNA; lane 5, M. snqmaris NCTC 8 159 DNA; lane 6, M. smegmaris mc’155 DNA. (b) Isolation of DNA from 1 mg to 30 mg (wet weight) of M. smegmatis (NCTC 8159). Lane I, molecular mass markers: lane 2, DNA isolated from 1 mg of cells; lane 3, DNA isolated from 2 mg of cells; lane.4, DNA isolated from 3 mg of cells; lane 5, DNA isolated from IO mg of cells: lane 6, DNA isolated from 30 mg of cells. In each case 5 ~1 of a total volume of 20 /*I was loaded onto the gel
from approx 1 X 109(1 mg wet weight of cells) to 3 X lO”‘(30 mg wet weight of cells) cells (see Fig. 2b). The product obtained was approx. 23 kbp in size and an apparently higher molecular mass fraction was always present (see Fig. 2a). The yield of purified DNA (approx. 2.5%) is similar to that obtained previously [9,11]. The DNA samples were found to be essentially free from DNAase activity because there was no change in the electrophoretic pattern (cf Fig. 2a) after incubation in the buffer used for the restriction endonuclease &I, at 37°C for 16 h. Also, as shown below, the DNAs were suitable substrates for restriction endonucleases and for amplification by PCR. 3.2. Restriction fragments
qf rRNA
(t-m) operons
Genomic DNAs were isolated as described in Fig. 1 and the number of rrn operons per genome was determined by revealing the number of PstI fragments having 16s rRNA gene sequences as described in Fig. 3. The results show that M. neoaurum has two rrn operons per genome, like M. ,fortuitum, M. phlei and M. smegmatis; whereas M. tuberculosis has a single rrn operon [ 18,191. In each case a PstI fragment of 1.2-1.4 kbp was identified.
3.3. Amplification
by PCR
fragments of M. smegmatis and M. of 1373 bp and 1267 bp respectively provide a frame of reference for the study of the rrn The
PsrI
tuberculosis
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JKLM
kb -23.1 -3.2
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_ --
- ._ ” _.___..
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Fig. 3. Investigation of the number of rm operons per genome. Genomic DNA was restricted with PsrI, the fragments were separated, transferred to a nylon membrane and prubed with a “P-1abelled 16s rRNA gene sequence (positions I-3551 as described in Materials and methods. Lane A, M. neoclwmm DNA; lane B, sample restricted with &I. Lane C. M. ,forluitum DNA; lane D. sample restricted with /%I. Lane E, M. phki DNA; lane F, sample restricted with PstI. Lane G, M. srneKnzuris (NCTC 8159) DNA; lane H, sample restricted with &I. Lane I, M. .smegmatis (mc’l55) DNA; lane J, sample restricted with &I. Lanes K and L, no DNA. Lane M, M. tuberculosis DNA restricted with WI.
J.A. Gon,-alrr-~-Mer~hund et ~11. / FEMS Microbiology Letters 135 (IYY6) 71-77
76
Py
100 basepairs
I
JY13-
approx 1025 bp
4
)f-
approx 296 bp ---_)
approx 1320 bp
_)
Fig. 4. Region
of an operon of the rrn A family upstream from the 16s rRNA gene. The location and size of the shown. JG7 + etc., binding sites for the primers used in PCR amplification: the sizes of the products obtained primers are indicated.
operons of other mycobacterial species because their nucleotide sequences have been established. The EMBL accession numbers for the nucleotide sequence data are X87944 (M. tuberculosis, see also [14]) and X87943 (M. srnegmatis). The two PstI fragments are homologous: each fragment has the 3’-terminal 114 codons of an open reading frame which is significantly similar to a gene for UDP-Nacetylglucosamine 1-carboxyvinyl transferase (EC 2.5.1.7) at its S-end and each extends downstream to include the first approx 655 bp of the 16s rRNA gene (see Fig. 4). The binding sites for primer JG7 kb
PstI fragment studied is for a particular
pair of
are located at the 5’-ends of the above mentioned PstI fragments and the binding sites for primer RAC8 lie within the 16s rRNA gene approx. 296 bp upstream from the 3’-PsrI site. Thus primers JG7/RAC8 should amplify homologues of the 1,373 bp PstI fragment of the rrn A, operon of M. smegmatis less 296 bp of the 16s rRNA gene [ 121. The results presented in Fig. 5a reveal that a homologue of the M. smegmatis PstI fragment, is present in each of the genomes studied; the sizes of the fragments lie in the range 0.95- 1.1 kbp. The rrn A, operon of il4. smegmatis and the rrn
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2.07 1.5 ---_,
0.6
0.6 -
(4
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Fig. 5. Amplification by PCR of regions of the rrn operons of mycobacteria. (a) Products obtained by amplifying the region from the S-&I site to position approx. 355 of the 16s rRNA gene. Lane 1, molecular mass markers; Lanes 2 and 8, no DNA; Lane 3, M. ne~aurum DNA; Lane 4, M. forruirum DNA; Lane 5, M. phlei DNA; Lane 6, M. smegmatis (NCTC 8159) DNA; Lane 7, M. smegmtis (mc’ 155) DNA; Lane 9, M. tuberculosis DNA. The faster moving bands in lane 3 are an artefact unrelated to rrn operons. (b) Products obtained by amplifying the region from approx 200 bp upstream from the 5’.end of the 16s rRNA coding region to position approx. 355 of the 16s rRNA gene. Lane 1, molecular mass markers; Lanes 2 and 3, no DNA: Lane 4, M. neoaurum DNA; Lane 5, M. fortuitum DNA; Lane 6, M. phlei DNA; Lane 7, M. smrgmatis (NCTC 8159) DNA; Lane 8, M. smqmaris (mc’ 155) DNA; Lane 9, M. tuberculosis DNA.