Molecular Methods for the Detection of Methanotrophs Ian R. McDonald, Andrew J. Holmes, Elizabeth M. Kenna, and J. Colin Murrell 1. Introduction Methane-oxidizmg bacteria (methanotrophs) are a unique group of bacteria that grow on methane as then sole source of carbon and energy. They can be isolated from a wide variety of marme and freshwater environments, soils, and sediments and appear to be ubiquitous in the natural environment. They have been classtfred, on the basis of chemotaxonomic studies and 16s rrbosomal RNA phylogenetic analyses, mto five genera: Methylococcus, Methylobacter, Methylomonas, Methylosmus, and Methylocystis (1,2). These five genera fall mto two phylogenetically distinct, exclusively methanotrophic groups. Methanotrophs with type I mtracellular membranes include the genera Methylomonas, Methylobacter, and Methylococcus, which are all related to bac- terra of the y-subdivision of the Proteobacterza. Methanotrophs with type II membranes include Methylosinus and Methylocystis, which belong to the a-subdivision of the Proteobacterla (Table 1). There has been considerable interest in methanotrophs since it has been recognized that they are a major smk for atmospheric methane In many natural environments, where these aerobic bacteria are exposed to methane, arising from the biological production by methanogens, they are responsible for removal of much of this methane before rt escapes to the atmosphere, and are therefore important in the global carbon cycle (see ref. 3 and chapters therein). The methanotrophs have also attracted considerable attention since they are able to degrade a number of important ground-water pollutants, such as trrchloroethylene (TCEtZ) and other halogenated hydrocarbons (3 and chapters therein). The enzyme responsible for biodegradation of TCEtl and other pollu- From Methods ~1 Wotechnology, Vol 2 B/oremed/atfon Protocols Edlted by D Sheehan Humana Press Inc , Totowa, NJ 111
Transcript
Molecular Methods for the Detection of Methanotrophs
Ian R. McDonald, Andrew J. Holmes, Elizabeth M. Kenna, and J. Colin Murrell
1. Introduction Methane-oxidizmg bacteria (methanotrophs) are a unique group of bacteria
that grow on methane as then sole source of carbon and energy. They can be isolated from a wide variety of marme and freshwater environments, soils, and sediments and appear to be ubiquitous in the natural environment. They have been classtfred, on the basis of chemotaxonomic studies and 16s rrbosomal RNA phylogenetic analyses, mto five genera: Methylococcus, Methylobacter, Methylomonas, Methylosmus, and Methylocystis (1,2). These five genera fall mto two phylogenetically distinct, exclusively methanotrophic groups. Methanotrophs with type I mtracellular membranes include the genera Methylomonas, Methylobacter, and Methylococcus, which are all related to bac- terra of the y-subdivision of the Proteobacterza. Methanotrophs with type II membranes include Methylosinus and Methylocystis, which belong to the a-subdivision of the Proteobacterla (Table 1). There has been considerable interest in methanotrophs since it has been recognized that they are a major smk for atmospheric methane In many natural environments, where these aerobic bacteria are exposed to methane, arising from the biological production by methanogens, they are responsible for removal of much of this methane before rt escapes to the atmosphere, and are therefore important in the global carbon cycle (see ref. 3 and chapters therein).
The methanotrophs have also attracted considerable attention since they are able to degrade a number of important ground-water pollutants, such as trrchloroethylene (TCEtZ) and other halogenated hydrocarbons (3 and chapters therein). The enzyme responsible for biodegradation of TCEtl and other pollu-
From Methods ~1 Wotechnology, Vol 2 B/oremed/atfon Protocols Edlted by D Sheehan Humana Press Inc , Totowa, NJ
111
Table
1
Cla
ssific
atio
n of
Met
hano
troph
sa
Met
hylom
onas
M
ethy
loba
cter
M
ethy
loco
ccus
M
erhy
losm
us
Met
hylo
cyst
ls
No.
of s
peci
es
3 6
2 2
2 Ph
ylog
eny
y Pr
oteo
bact
ena
y Pr
oteo
bact
ena
y Pr
oteo
bact
ena
a Pr
oteo
bact
ena
a Pr
oteo
bact
ena
Mem
bran
e ty
pe
Type
I
Type
I
Type
I
Type
11
Type
II
Fatty
ac
id
16 1
16
:l 16
.1
18.1
18
:l P
athw
ay fo
r ca
rbon
as
sim
ilatio
n R
UM
P
RU
MP
R
uMP
/Sen
ne
Sen
ne
Sen
ne
MM
0 en
zym
e ty
pe
pMM
0 pM
M0
pMM
O/s
MM
O
pMM
O/s
MM
O
pMM
O/s
MM
O
Mol
%G
+C
cont
ent o
f DN
A
50 -5
4 50
-54
62.5
62
-63
62-6
3 N
2 fix
atio
n V
+ +
+ C
arot
enol
ds
+ M
arin
e re
pres
enta
tives
-
+ -
“RUM
P,
R&do
se
Mon
opho
spha
te
Path
way
, pM
M0,
par
ticul
ate m
etha
ne mon
ooxy
gena
se, sM
M0,
sol
uble
met
hane
mon
ooxy
gena
se
Detection of Methanotrophs 113
tants IS methane mono-oxygenase (MMO), whose normal role m these organ- isms is the conversion of methane to methanol. Two forms of MM0 are known, a cytoplasmic (soluble MMO) and a membrane-associated form (particulate MMO). The enzyme that has been studied in most detail is the soluble MM0 (sMMO), for which detailed brochemical and genetic mformatron exists (for review see refs. 3,4). The wide range of substrates cooxrdrsed by sMM0 make it an important enzyme for the study of breakdown of a wide variety of chlon- nated alkanes, alkenes, and aromatic compounds. The range of substrates coox- idrsed by pMM0 is more restricted, although it still includes some rmportant pollutants (e.g., TCE). This form of the enzyme is universally present m all methanotrophs. The genes encoding the sMM0 enzyme complex (mmo genes) have been extensively characterized in two methanotrophs (4). They show a high level of homology which makes them useful as functional gene probes for the detection of sMMO-containing methanotrophs m natural environmental samples and in bioremediatron programs designed to exploit their cooxrdatton properties for the degradation of TCE and other pollutants (e.g., see ref. 5).
The sMM0 complex of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b contams three proteins. Protein A IS the hydroxylase com- ponent and consists of three polypeptrdes a, p, and y; Protein B IS a coupling protein and Protein C is the reductase component. They are coded for by the genes, mmoX, mmoY, mmoZ, mmoB, and mmoC, respectively, which are clus- tered on the chromosome of both of these (and other) methanotrophs (4).
Molecular ecology techniques may be used to detect the presence and iden- tity of methanotrophs directly in envn-onmental samples, including broremedr- atron sites. Techniques and strategies available for methanotrophs have recently been reviewed in detail (6). Nucleic acid probes available fall into two classes: (I) functional and (11) phylogenetic. The only functronal probes for methanotrophs presently available target sMM0 genes and are subsequently limited for use with methanotrophs that possess this form of the enzyme m addition to the pMM0 (7). Probes targeting pMM0 (8) genes are presently under development in the authors’ laboratory and would represent a umver- sally applicable methanotroph functional gene probe (4). Phylogenetic trees constructed from 16s rRNA sequences by Hanson, Bowman, and colleagues (2,9) have revealed exclusively methanotrophrc clusters. This makes tt possi- ble to design phylogenetic group-specific (16s rRNA) probes for methan- otrophs targeting the 16s rRNA. Both classes of probe can now be used directly to detect methanotrophs in a variety of environments, obviating the sometimes difficult task of enriching for and isolatmg them. The followmg methods describe the polymerase chain reaction (PCR) amplification, detec- tion, and characterrzatron of methanotroph-specific sMM0 and 16s rRNA genes in natural environmental samples
114 McDonald et al.
2. Materials
2.1. Bacterial Strains and Reagents
All bacterial strains described here can be obtained from the University of Warwick Culture Collection and the NCIMB (Aberdeen, Scotland). Control organisms used in PCR expertments: Ms. trichosponum OB3b, MC. capsulatus (Bath), Methylosinus sporzum 5, Methylocystis strain M (all sMMO+), Methylocystis parvus OBBP, Methylobacter agile A20, Methylomonas methanlca S 1, Methylobacter albus BG8 (all sMMO-), Methylobacterzum extorquens AM1 (sMMO-, methanol dehydrogenase (MDH)+), Escherichza coEi DHl (sMMO-, MDH-). The E. colt host used for cloning experiments IS E coli DHl or, where stated, commercially available E.coli strains. Chromosomal DNA is prepared from methanotrophs by the method of Oakley and Murrell (10) although any standard molecular biology laboratory procedure should work well for these organisms.
Oligonucleotide primers are synthesized on a PE Applied Biosystems (Warrmgton, Cheshire, UK) DNA synthesizer. (Many commercial outlets now exist for the custom-made synthesis of oligonucleottdes). Fluorescently labeled oligonucleotide probes were obtained from Genosys (Cambridge, UK). All reagents used are Molecular Biology grade (where available) or Analar grade (Sigma, Poole, Dorset, UK). Good quality double-drstilled, deionized water should be used for preparation of all solutions. Taq DNA polymerase and poly- merase buffer are obtamed from Gibco-BRL (Paisley, Scotland). Mineral oil is from Sigma. Nylon membrane is from Amersham (High Wycombe, UK). The TA cloning kit for clonmg of PCR products is available from Invitrogen (San Diego, CA). The Sequenase Version 2.0 Sequencing Kit is available from United States Btochemicals (Cleveland, OH). The DNA thermal cycler used in these methods 1s the Combi Thermal Reactor TR2 (Hybaid, Teddington, Middlesex, UK). Hybridizations are carried out m an Oven (Hybaid). T4 polynucleotide kmase, T4 kmase buffer, and DNA polymerase for nick translation are available from Gibco-BRL. All radiolabels (T-‘~P ATP and 32P dGTP) are obtained from Amersham (High Wycombe, UK).
6. Oligonucleotlde hybridization buffer contains 6X SSC, 0.5% SDS, 10 mM sodium phosphate, pH 6.8, 200 pg/mL denatured Herring testes DNA (Sigma, UK), 5X Denhardt’s solution The Herring testes DNA solution is denatured by boiling for 20 mm. This hybridization buffer 1s freshly made before use.
7 Southern blot hybrldlzatlon buffer contains 6X SSC, 0 5% SDS, 200 pg mL dena- tured Herring testes DNA (Sigma), 5X Denhardt’s solution. The Herring testes DNA solution 1s denatured by boiling for 20 min (freshly made before use)
9 Phosphate-buffer saline (PBS) 390 mM NaCI, 30 mM, phosphate buffer, pH 7 2 10. Hybrldlzatlon solution for in situ hybridization: 900 mM NaCl, 20% formamlde,
0.01% SDS, 20 mM Tns-HCl, pH 7 2.
3. Method
3.1. Total DNA Extraction from Environmental Samples 3.7.7. DNA Extractrons from Water Samples
DNA from water samples (e.g. river water, pond water, seawater) may be extracted using a method essentially as described by Sommerville et al. (II). In our laboratory Sterivex 0.22~ym filter units (Mlllipore, cat. no. SVGSO1015) are used, although any slmllar self-contamed filtration apparatus could be used. The volume of water sample that may be passed through these units varies from 250 mL to 1 L, depending on the particle load of the water sample. Samples can easily be processed through the filters at the collection site using a sterile, large volume syringe, frozen, and then transported to the laboratory for further work. The following describes the protocol employed.
1. Collect bacterial cells on the filter by aseptically passing 250-1000 mL of water through the Sterlvex unit
2. Wash the cells by passing 10 mL sterile SET buffer through the unit Excess buffer 1s removed by pushing through air from an empty syringe. The filter units are then capped to prevent contammation and should be frozen at -20°C if they are not to be processed immediately (see Note 1)
3 Using a 25-gage needle, add 1 8 mL SET buffer through the inlet of the filter unit (longer needles may puncture the filter) Thirty mlcrohters of lysozyme (10 mg/mL) 1s then added and the inlet recapped Invert the filter unit 3-4 times to mix the contents and incubate at 4°C for 15 mm with occasional mixing.
4. Twenty microliters of a 20% SDS solution 1s added and the filter incubated at room temperature under constant rotation for 1 h. A Ummlx 380 (Luckham Ltd., Sussex, UK) 1s used to roll the filter umts (see Note 2) At the end of this treatment, 50 pL of Proteinase K (20 mg/mL) 1s added and the filter units incubated on the roller for a further 3 h
116 McDonald et al
5 The crude lysate 1s harvested by wtthdrawmg it back through the filter unit inlet with a 5 mL syringe The filter IS washed by addmon of 1 mL SET buffer to the unit and replacing tt on the roller for 5 mm The wash buffer IS collected as above and pooled with the crude lysate.
6. Add 0 5 vol of 7 5M ammomum acetate and mix by inverting the tube 34 times Incubate the mix at room temperature for 15 mm and then centrifuge at 12,OOOg for 5 min to pellet dissociated proteins. Transfer the supernatant to a new tube and pre- cipitate nucleic acids by addmon of 2 5 vol ethanol The tubes are incubated at -20°C for 30 mm then centrifuged at 12,000g for 20 mm to pellet the nucleic acids. The pellet IS washed by addition of 500 pL 70% ethanol and centrtfugatton for 5 mm The supernatant IS carefully removed with a ptpet tip attached to a vacuum lme and the pellet 1s an-dried. The nucleic acid pellet IS then resuspended m 300 pL sterile water and the ammomum acetate treatment IS repeated.
This crude puriftcatron procedure yields high-mol-wt DNA (typically 20-30 pg/L of water sample) of sufftcient purity, for amplrfication of mmo and 16s rRNA genes by the PCR, with estuarme, coastal seawater, open ocean marine samples, pond, lake, and riverwater samples (see Note 3)
3.1.2 DNA Extractions from So11 and Sediment Samples
Total DNA isolation from fresh soil, sediment, and peat samples is essen- tially as described by Selenska and Klingmuller (12) and Bruce et al. (13).
1 Suspend the sample (2 g wet weight) in 5 mL of extraction buffer (1% SDS m 0 12M Na2HP04, pH 8.0) and incubate at 70°C for 1 h with occasional shakmg (see Note 4)
2. Centrifuged the sample at 2000g for 10 mm and hold the supernatant at 4°C 3 Extract twice more with 5 mL of fresh extraction buffer. The three supernatant frac-
tions are pooled and centrifuged at SOOOg for 30 mm. 4. Add polyethylene glycol (PEG) to the supernatant to a final concentration of 15%
(15 mL of a stock solutton of 30% PEG 6000 m TE buffer, pH 8 0); 1.5 mL of 5M NaCl 1s also added gradually and shaken.
5 Precipitate overnight at 4°C and centrifuge at 5OOOg for 20 mm. Resuspend the pel- let m 4 mL of TE, gtvmg a brown solution to which 4 g of CsCl and 100 pL of ethrdmm bromide (EtBr)( 10 mg/mL) are also added
6 Remove the DNA band after ultracentrifugatton at 290,000 g overnight m a Sorvall Vti 65 rotor (see Note 5), extract the EtBr with TE saturated 1-butanol, and dialyze overnight in TE to remove the CsCl.
7. Precipitate the DNA with ammomum acetate and ethanol (1 vol DNA solution, l/3 vol of 10.5M ammomum acetate and 2 vol of ethanol) at -20°C overnight and wash m 70% ethanol to remove any remaining brown color (see Note 6)
8. Resuspend the DNA m 400 pL of TE.
Good quality high-mol-wt DNA, with typical yields of 100 pg DNA/2 g sam- ple, 1s obtained and is suitable for PCR of mmo and 16s t-RNA genes (see Note 7)
Detect/on of Methanotrophs 117
3.2. PCR-Amplification of Methanotroph Genes from Environmental DNA Samples
3.2.1. Phylogenetic Group-Speclfrc Pnmers
16s rRNA sequences of methanotrophs can be obtamed from Genbank and Bowman (9). Regions diagnostic for each of the representatives of the five gen- era of methanotrophs have been selected for probe design (see Note 8) These 16s rRNA probes and their control organisms are shown m Table 2. They have been tested for their ability to detect methanotroph genera specifically m both PCR and colony hybridizations against cloned 16s rRNA genes from represen- tative methanotrophs m the University of Warwick culture collectton. Reaction conditions for hybridization and PCR testing are described m the following sec- tions. All probes give a strong signal with their target genes and show no cross- reactivity with methanotrophs from other genera (see Note 9).
3.2.2. “‘Functional Group-Specific” Primers
“Functional group-specrftc” probes may be defined as those that target genes that are specific for a particular cell activity. Where the cell acttvrty defines a microbial physiological group, such probes may be termed “func- tional group-specific”. We have selected genes encoding two enzymes as tar- gets for functional group-specific probes; mxuF, encoding the large subunit of methanol dehydrogenase (MDH); and the genes encoding sMM0 (mmoX, mmoY, mmoZ, mmoB, and mmoC). mxaF is expected to to be a methylotroph- specific probe capable of detecting all Gram-negative methylotrophs, mclud- mg the methanotrophs. The mmo probes are methanotroph-specific but will detect only the sMMO+ methanotrophs and not those methanotrophs which contain only the pMM0.
The mmo primers were designed from the published sequences of the sMM0 gene clusters of Methylococcus capsulatus (Bath) and Methylosmus tnchospo- rium OB3b (4 and references therein). mxaF primers were based on published sequences of three species of methylotrophs (15). These primers amplify then target fragments spectfrcally from all methanotrophs in our collection (mxaF primers) and all sMMO+ strains (mmo primers) (see Note 10). The primer sequences are shown m Table 2.
3.2.3. Reaction Conditions for PCR
Table 3 shows typical reaction components for a PCR. The concentratton of MgC12 and template DNA varies according to the source of the DNA. The fol- lowing protocol describes reaction mixture and amplification conditions used for control DNA samples extracted from pure cultures.
Table
2
Met
hano
troph
So
lubl
e M
etha
ne
Mon
ooxy
gena
se
(sM
M0)
an
d Ph
yloge
netic
G
roup
-Spe
cific
Prob
es/P
rimer
s
Col
ony
Con
trol
hybn
drza
tron
PCR
Pnm
er
Sequ
ence
(S-
3’)
Targ
et
genu
s or
gani
sm
tem
pera
ture
te
mpe
ratu
re
Mb
1007
r C
ACTC
TAC
GAT
CTC
TCAC
AG
Met
hylo
bact
er
Mb
albu
s B
G8
60°C
58
°C
MC
100
%
CC
GC
ATC
TCTG
CA
GG
AT
Met
hylo
cocc
us
MC
cups
ulat
us
Bat
h 55
°C
54°C
M
m
1007
r C
AC
TCC
GC
TATC
TCTA
AC
AG
M
ethy
lom
onas
M
m.
met
ham
ca
S 1
55°C
58
°C
MS
102
0r
CC
CTT
GC
GG
AA
GG
AA
GTC
M
ethy
losc
nus
MS
tnch
ospo
rzum
O
B3b
55
°C
54°C
m
xaF
1003
f G
CG
GC
AC
CA
AC
TGG
GG
CTG
GT
All
Gra
m-n
egat
we
Met
hylo
troph
s M
S trz
chos
porz
um
OB
3b
Not
tes
ted
59°C
w
rxaF
156
1r
GG
GC
AG
CA
TGA
AG
GG
CTC
CC
A
ll G
ram
-neg
ativ
e M
ethy
lotro
phs
MS
trzch
ospo
rzum
O
B3b
N
ot t
este
d 59
°C
mm
oX
882f
G
GC
TCC
AA
GTT
CA
AG
GTC
GA
GC
sM
MO
+ m
etha
notr
ophs
M
S trz
chos
porz
um
OB
3b
Not
tes
ted
55°C
m
moX
14
03r
TGG
CA
CTC
GTA
GC
GC
TCC
GG
CTC
G
sMM
O+
met
hano
trop
hs
MS
tnch
ospo
num
O
B3b
N
ot t
este
d 55
°C
mm
oY
198f
C
CG
AC
TGG
ATC
GC
CG
GC
GG
CC
T sM
MO
+ m
etha
notr
ophs
M
S tn
chos
porz
um
OB
3b
Not
tes
ted
37°C
mm
oY
8201
C
GC
TGG
AAG
AAC
TCG
CG
GC
GG
sM
M0+
met
hano
troph
s sM
MO
+ m
etba
notro
phs
sMM
O+
met
hano
troph
s sM
MO
+ m
etba
notro
phs
sMM
O+
met
hano
troph
s sM
MO
+ m
etha
notro
phs
sMM
O+
met
hano
troph
s A
ll Eu
bact
ena
All
Euba
cten
a M
ethy
lom
onas
MS
tnch
ospo
num
O
B3b
Not
te
sted
37
°C
mm
oZ
133f
C
GC
CG
TTC
CG
CAA
GAG
CTA
CG
A M
s. t
ncho
spon
um
OB3
b N
ot
test
ed
55°C
m
moZ
483
r TT
GC
GC
AGC
CC
TTC
CAG
CG
GC
GTG
M
s. t
ncho
spor
ium
O
B3b
Not
te
sted
55
°C
mm
oB 7
7f
AGTT
CTT
CG
CC
GAG
GAG
AAC
CA
Ms.
tnc
hosp
onum
O
B3b
Not
te
sted
55
°C
mm
oB 3
69r
TGC
CC
AGG
GTG
TAG
GC
GC
GG
CC
GA
MS
tnch
ospo
num
O
B3b
Not
te
sted
55
°C
mm
oC 5
42f
GG
TTC
TGC
TGTG
CC
GC
ACC
M
S tn
chos
ponu
m
OB3
b N
ot
test
ed
55°C
m
moC
986
r AT
CCCG
TGCC
GCC
GG
CGAC
G
MS
tnch
ospo
num
O
B3b
E. c
olt
E. c
o11
Mm
. m
etha
nlca
S
1
Not
te
sted
N
ot t
este
d N
ot t
este
d N
ot t
este
d
55°C
55
°C
55°C
58
°C
16s
rRNA
27
f AG
AGTT
TGAT
CM
TGG
CTC
AG
16s
rRNA
14
92r
TAC
GG
YTAC
C’IT
GTT
ACG
ACTT
M
m
850
TAC
GTT
AGC
TCC
ACC
ACTA
A
120
Table 3 Typical Reaction Components for PCR
McDonald et al
Reaction component Concentration m PCR
Polymerase buffer (Gtbco-BRL)
W32
W-l detergent (Grbco-BRL) Prtmer 1 Primer 2 DNA template dNTP (concentratron of each nucleotrde) Taq polymerase (Gtbco-BRL)
20 mM Trrs-HCl, 50 mM KC1 25mM 005% 0 l-l @!! 0.1-l @I 10 ng-10 yglmL 200 p.M 25u
Prepare a master mix (see Note 11) containing reaction buffer, MgC12 (see Note 12), BSA, and each deoxynucleotrde and ahquot into 0 7-mL mtcrocentrtfuge tubes. Add 100 pmol of each pnmer to each tube Add 10 ng of template DNA to each tube (see Note 13) Vortex tubes briefly to mix components and give a pulse spm m a mrcrocentrrfuge Overlay the reaction components with 50 pL mineral or1 Place the tubes in the thermal cycler and heat to 94°C for 5 mm to denature the tem- plate DNA During this time a fresh dilution (1 10) of stock Tuq polymerase (5 U/pL) is prepared m sterile drstrlled water A “hot start” PCR is employed by paus- mg the thermal cycler at 92°C and adding 5 pL (2 5 U) of the diluted Tuq poly- merase through the oil layer mto the aqueous phase Thirty cycles of annealing, extension, and melting are then carried out as below; 55°C for 1 mm, 72°C for 1 mm, 92°C for 1 min This IS followed by a final cycle of 55°C for 1 mm, 72°C for 5 mm The reactions are stored at -20°C until analyzed Take a 5-pL ahquot of the reaction for visual exammatton by agarose gel elec- trophoresrs
Examples of typical PCR results for control DNAs are shown m Fig. 1 and with envr- ronmental DNA samples m Rg 2 All sets of PCRs should contain both a negative (1 e , a tube with all reagents but with no template) and a positive (i.e., a tube with a known DNA sample of established punty) control
3.2.4. ConfirmIng Identity of PCR Products by Southern Hybndlzatlon
Amplifrcatron of the intended product can be confirmed by Southern blotting DNA from agarose gels onto nylon membrane and hybridizing this DNA with an appropriate probe (an oligonucleotlde or a DNA fragment; see Note 14), as outlined below.
Detection of Methanotrophs
2027bp-
564bp-
Fig. 1. PCR amplification of M. trichospotium OB3b DNA using methanotroph- specific primers. Lane 1 contains h Hind III size marker (Gibco-BRL); the following lanes contain the PCR products from amplification of M. trichosporium OB3b template DNA using the methanotroph-specific primers: Lane 2, mmoX, Lane 3, mmoY; Lane 4, mmoZ; Lane 5, mmoB; Lane 6, mmoC, Lane 7, moxp, Lane 8, 16s rRNA.
1. Load and run a 0.8% agarose (higher concentrations of agarose can retard transfer of DNA), 1X TBE gel.
2. Soak the gel for 2 x 20 min in denaturing solution (1.5M NaCl, OSM NaOH), fol- lowed by 2 x 20 min in neutralizing solution (IM Tris-HCl, pH 7.4, 1.5M NaCl).
3. Set up the Southern blot (16) and blot overnight to ensure complete transfer. 4. Fix DNA to the nylon membrane either by UV crosslinking or by baking the mem-
brane at 80°C for 2 h. 5. Prehybridize the membrane, with the appropriate buffer (20-50 mL)(see Section
2.), for 1 h in a Hybaid hybridization oven (up to six filters can be placed in each tube, sandwiched between nylon mesh).
6. The labeled probe (a nick-translated DNA probe or an end-labeled oligonu- cleotide probe) is denatured by boiling for 10 min and then added to the hybridiza- tion tube. a. Nick translation of probe: 250 ng DNA, 2X nick translation buffer (see
Section 2.), 3 pL unlabeled dNTPs (a solution containing three of the four dNTPs, each at a concentration of 5 mM), 10 pCi of c&~‘P dNTP, 10 U of DNA pol I, 1 pL DNase I solution (1 pL of 20 pg /mL stock diluted in 50 pL of water, then 1 pL of this into 50 mL of water), to a total volume of 20 pL with water. Incubate for 4 h at 4°C.
b. End labeling of probe: 500 ng DNA, 1X T4 kinase buffer, 10 U of T4 kinase, 50 pCi f2P-ATP, made up to 50 pL with water. Incubate at 37°C for 1 h.
7. The membranes are hybridized overnight at 65°C (or 50°C for the oligonucleotide probe).
8. Membranes are washed twice with 100 mL of 2X SSC at 80°C for 30 min (or with 6X SSC at 50°C for the oligo probe).
9. The filters are then air-dried and subjected to standard autoradiography techniques.
McDonald et al.
Fig. 2. PCR amplification of DNA extracted from environmental samples using mmoX-specific primers. Lane 1 contains h Hind111 size marker (Gibco-BRL): The fol- lowing lanes contain the PCR product from amplification with mmoX-specific primers of DNA extracted from environmental samples: Lane 2, freshwater; Lane 3, estuarine; Lane 4, sediment; Lane 5, soil; Lane 6, peat; Lane 7, M. trichosporium OB3b control.
3.3. Whole Cell Approaches
3.3.1. Probes for ‘In Situ’ Hybridization
Oligonuclotide probes were obtained from Genosys (Cambridge, UK). Rhodamine or fluorescein moieties were directly incorporated during synthesis at the 5’ terminus using a C6 aminolinker. The probe Eub 338 (18) is used as a posi- tive control in all hybridizations. Probe Mm850 labeled with rhodamine has been used for the specific detection of Methylomonas sp. (Table 2; see Notes 15-17).
3.3.2. Fluorescent ‘In Situ’ Hybridization
The procedure given below is used for the identification of methanotrophs in cultures:
1. Collect 1 mL of a fresh culture (see Note 18) into a 5 mL bijou bottle and add 3 mL of 4% paraformaldehyde (in PBS, pH 7.2). Incubate for a minimum of 3 h at 4°C. The fixed cells can be stored in 50% ethanol for at least 1 mo at -20°C.
2. Mix together in a 1.5 mL microcentrifuge tube, 100 p.L fixed cells and 10 p.L 1% Triton X-100. Centrifuge the mixture at low speed (6000g) to collect the cells and remove the supernatant. Resuspend the cell pellet in 10-20 pL of 0.1% Triton X- 100 to give a final concentration of 108-lo9 cells/ml.
3. Apply 3 pL of the washed, fixed cells to a gelatin-treated microscope slide in a spot approx l-2 mm2 (2 x 1.5 pL applications). Allow to dry. (Gelatin treated slides are made by cleaning microscope slides in a 10% ethanolic KOH solution followed by a rinse in distilled water. The slides are then dipped in a solution of 0.1% gelatin, 0.01% potassium chromate at 70°C and allowed to dry).
Detection of Methanotrophs 123
4. The cells are dehydrated by immersing the slide m a graded ethanol series (50, 80, 96%) for 2-3 mm each. The slides are then allowed to air dry
5 Add 10 l.tL Hybridization solutron (9 yL Hybridization buffer (see Section 2 and Note 19) + 50 ng ohgonucleotide probe) to the dried cell, smear, and incubate the slide m an isotonically-equilibrated humidrfied chamber (see Note 20) at an appro- priate temperature for 2-3 h.
6 Wash off the hybridrzatron solution with 1 mL of wash buffer to remove excess probe. Immerse the slides m prewarmed wash buffer at an appropriate temperature for 30 mm
7. Dip the slides m distilled water (1-2 s) to rmse off wash buffer and then allow to au dry. Apply 20 j.rL Citifluor AF3 to the smear and examine under a covershp on a Zeiss Axioskop microscope fitted with filter sets 09 and 15. (Typical results with this technique can be seen in Holmes et al (20)
3.4. Concluding Remarks
The choice of method used depends on the aim of the experiments. Nucleic acid-based techniques offer great sensitivity but only a semtquantitatrve capac- ity. Whole-cell-based approaches are less sensitive, but give better quantttatton. Both strategies are greatly affected by the choice of probe. The greatest database for probe design exists for the 16s rRNA. Because this is a universally present gene, interpretation of results from phylogenetic group-specific probes target- ing this must be cautious. The sMM0 is thought to be unique to methanotrophs, however, not all methanotrophs have this gene and the small database means that the present generatron of probes must be carefully controlled. Recommendations for which sMM0 gene prrmers to use to detect mmo genes are given in McDonald et al. (7), however, we suggest that primers for mmoY are not used because the optimum temperature for PCR was 37°C and the use of mmoX primers probably give the most reliable indication of the presence of the soluble MM0 gene. The pMM0 is found in all methanotrophs but is also similar to the ammonia monooxygenase (AMO) of the nitrrfying bacteria. Both pMM0 and AM0 have a similar substrate range, so it may be useful to detect both; however, the small database makes methanotroph-specific probes impos- sible to design at present.
4. Notes
1. Convenient caps can be made by sealing the ends of 1-mL prpet tips m a bunsen flame and then trimming the tip to fit the Sterivex unit outlet A sterile syringe IS used as the cap for the mlet
2. Sommervtlle et al (II) report a design for a homemade roller. 3 In some cases, the DNA 1s resrstant to amplificatron. Additional purtfrcation by
CsCl gradient centrrfugation, as described from step 5 onward below, resolves thus problem
McDonald et al
4
5
6
7
8
9
10.
Il.
12
13
14
15
It 1s important to ensure that the soil sample always remams suspended m the extraction buffer; vortexmg is often required If no bands are seen after ultracentrtfugatton, tubes can be centrifuged again and bands are often then produced Not all the brown color may be removed but DNA ~111 be sufficiently clean for PCR after two or three washes with 70% ethanol If excessive degradation of the DNA is observed, difficulty ~111 be experienced m the amphfication of large products and the incidence of chimera formatton may be htgh (14). Almost 3000 16s rRNA sequences from bacteria are presently available m the ribo- somal database project (RDP) This represents an invaluable resource for probe design but is probably >l% of the total bacterial diversity Probes targeting 16s rRNA cannot be guaranteed to be uniquely specific to a target group because it is impossible to test this at present The Methylococcus-specific probe descrrbed here has been found to crossreact with umdentified nonmethanotroph clones in a soil gene library (Kenna, unpublished data) This probe cannot be used spectftcally to detect MethyEococcus by Itself but must be used m conlunctton with a second probe Primers targetmg protem-coding genes must cater for redundancy m the genetic code It 1s not possible to assume specifictty of the primers described here for all sMM0 genes We are presently experimentmg with the use of “umversal” nucleo- side analogs as alternatives to the mcorporation of multiple degeneracies m primers It is more convenient to prepare a master mix if the same PCR is to be done on a large number of samples. This improves reproducibihty and reduces the chance of contamination The effect of Mg2+ on PCR can vary tremendously accordmg to the template source/purity and the DNA polymerase used For environmental DNA samples it may be necessary to use very high Mg*+ levels (up to 10 mM) or carry out addi- tional purtfication steps to remove contaminants that are reducing the available Mg*+. Some enzymes are less susceptible to vartattons m Mg2+ and we recommend trying several suppliers’ polymerases Strong, easily visible bands are obtained from pure cultures using from 1 pg-100 ng of DNA as template We recommend the higher amount of template for envt- ronmental samples since the incidence of the target organism 1s unknown and potential template mhibmon effects (because of to msufficient purity) are more readily detected The identity of PCR products can be further confirmed by sequencing PCR prod- ucts can be cloned using commercially-available kits (1 e , TA clonmg Kit, Invitrogen, San Diego, CA) DNA IS then prepared from clones using the mnnprep method of Saunders and Burke (17) and sequenced using standard sequencmg methods (16) Probes that target rRNA must target domains of the molecule that are m easily accessible portions of the rtbosome A list of ribosomal target sites successfully used is given m Amann et al. (19)
Detect/on of Methanotrophs 125
16. Enzyme-labeled probes can be used for more sensitive detection but require spe- cialized treatment of the cell wall
17 It is desirable to use probes with a T, of <60°C because cells may burst at higher temperatures
18 The signal strength can be greatly improved by subculturmg an ahquot of cells into fresh medium and premcubatmg for 6 h prior to cell fixation to stimulate growth and hence ribosome synthesis
19 Other nomomc detergents are also suitable. The mclusion of formamide m the hybridization buffer is optronal Formamide decreases duplex stability, allowmg improved specificity of hybridization at lower temperatures and may also have an effect on increasing accessibihty of the probe to its target site within the ribosome.
20. A cheap, effective chamber can be made from a pipet tip box with a lmmg of tissue paper soaked m hybridization buffer
Acknowledgments This work was supported financially by the NERC TIGER initiative,
BBSRC, AFRC, and EEC
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2 Brusseau, G A , Bulygma, E S , and Hanson, R. S (1994) Phylogenetic analysis and development of probes for differentiatmg methylotrophic bacteria. Appl Envwon Mlcroblol 60,626-636.
3 Murrell, J C , and Kelly, D P (1993) MicrobzaZ Growth on Cl Compounds, Intercept, Andover, UK.
4 Murrell, J C. (1994) Molecular genetics of methane oxidation. Biodegradation 5, 145-159
5 Alvarez-Cohen, L , McCarty, P L , Boulygma, E , Brusseau, G , and Hanson, R. S (1992) Cometabolic biotransformation of tnchloroethylene and chloroform by a bac- terial consortmm grown with methane. Appl Environ. Mzcrobzol. 58, 1886-1893.
6 Murrell, J C , Holmes, A J , McDonald, I. R., and Kenna, E (1995) The develop- ment of molecular ecology techniques for the detection of methanotrophs m the natural environment, m The Mzcrobiology of Atmospheric Trace Gases: Sources, Sznks and Global Change Processes, (Murrell, J C and Kelly, D P., eds ), NATO AS1 Series, Springer Verlag, pp 135-15 1
7 McDonald, I R., Kenna, E M , and Murrell, J C (1995) Detection of methan- atrophic bacteria m environmental samples by using the polymerase chain reaction. Appl Environ Microblol 61, 116-121
8 Semrau, J. D., Chlstoserdov, A., Lebron, J., Costello, A., Davagnmo, J., Kenna, E , Holmes, A J , Finch, R , Murrell, J. C , and Lidstrom, M E (1995)
9. Bowman, J P (1992) The systematics of methane-utihzmg bacteria PhD Thests, Umverstty of Queensland, Brisbane, Austraha
10. Oakley, C. J , and Murrell, J. C. (1988) ni$4 genes m obhgate methane oxtdtsmg bacteria FEMS Mlcroblol. Lett 49,53-57
11 Sommerville, C. J , Knight, I T , Straube, W L , and Colwell, R R. (1989) Simple, rapid method for direct isolation of nucleic acids from aquatic environments Appl Environ Mlcroblol 55,548-554
12. Selenska, S , and Klingmuller, W (1991) DNA recovery and direct detection of Tn5 sequences from so11 Lett Appl Mlcroblol 13,21-24
13 Bruce, K D , H~oms, W D , Hobman, J L. Osbom, A M , Stroke, P , and Ritchie, D A (1992) Amplification of DNA from native populations of soil bacteria by usmg the polymerase chain reaction. Appl Envzron Mlcrobzol. 58,3413-3416
14 Ltesack, W , Weyland, H , and Stackebrandt, E (1991) Potential risks of gene amphfication by PCR as determined by 16s rDNA analysis of a mtxed-culture of strict barophthc bacteria Mzcrob Ecol 21, 191-198
15. Murrell, J C , McGowan, V., and Cardy, D L N (1993) Detection of methy- lotrophic bacteria m natural samples by molecular probmg techniques Chemosphere 26, 1 - 11
16 Sambrook, J , Frnsch, E F , and Mamatis, T (1989) Transfer of DNA from agarose gels to solid supports, m Molecular Cloning A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory, Cold Sprmg Harbor, NY, p. 9.34.
17. Saunders, S E , and Burke, J F (1990) Rapid isolation of mmiprep DNA for dou- ble strand sequencing Nucleic Aczds Res l&4948
18 Amann, R I , Stromley, J , Devereaux, R , Key, R., and Stahl, D A (1992) Molecular and microscoptc identiftcatton of sulfate-reducmg bacteria m multi- species biofilms Appl Environ Microbzol 58,614-623
19 Amann, R I , Ludwig, W , and Schleifer, K H (1995) Phylogenettc tdentification and zn situ detection of mdividual mtcrobial cells without cultivation Mzcrobzol Rev 59,143-169
20 Holmes, A. J , Owens, N J P , and Murrell, J C (1995) Detection of novel marme methanotrophs using phylogenetic and functional gene probes after methane enrichment Mzcroblology 141,1947-1955
