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Previous Article | Next Article 
Applied and Environmental Microbiology, June 2001, p. 2538-2544, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2538-2544.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chromosomal Gene Inactivation in the Green Sulfur
Bacterium Chlorobium tepidum by Natural
Transformation
Niels-Ulrik
Frigaard* and
Donald A.
Bryant
Department of Biochemistry and Molecular
Biology, The Pennsylvania State University, University Park,
Pennsylvania
Received 12 December 2000/Accepted 18 March 2001
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ABSTRACT |
Conditions for inactivating chromosomal genes of Chlorobium
tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with
various antibiotic resistance markers. Growth of wild-type C.
tepidum at 40°C on agar plates could be completely inhibited
by 100 µg of gentamicin ml
1, 2 µg of erythromycin
ml1, 30 µg of chloramphenicol ml1, or 1 µg of tetracycline ml1 or a combination of 300 µg of
streptomycin ml1 and 150 µg of spectinomycin
ml1. Transformation was performed by spotting cells and
DNA on an agar plate for 10 to 20 h. Transformation frequencies on
the order of 107 were observed with gentamicin and
erythromycin markers, and transformation frequencies on the order of
103 were observed with a
streptomycin-spectinomycin marker. The frequency of spontaneous
mutants resistant to gentamicin, erythromycin, or
spectinomycin-streptomycin was undetectable or significantly lower than
the transformation frequency. Transformation with the gentamicin marker
was observed when the transforming DNA contained 1 or 3 kb of total
homologous flanking sequence but not when the transforming DNA
contained only 0.3 kb of homologous sequence. Linearized plasmids
transformed at least an order of magnitude better than circular
plasmids. This work forms a foundation for the systematic targeted
inactivation of genes in C. tepidum, whose 2.15-Mb
genome has recently been completely sequenced.
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INTRODUCTION |
Green sulfur bacteria are strictly
anaerobic phototrophs which occur in sulfide-rich aquatic environments
(21). They form a coherent phylogenetic group and are not
closely related to other bacteria (13). Current areas of
interest in this group of bacteria include photosynthetic electron
transport (involving the reaction center, cytochromes, quinones, etc.);
organization of and energy transfer in the light-harvesting antennae
(the FMO protein and chlorosomes); biosynthesis and function of
chlorophylls (bacteriochlorophylls a, c,
d, and e and chlorophyll a),
carotenoids, and isoprenoid quinones; lithotrophic oxidation of sulfur
compounds; CO2 fixation (which occurs via the
reverse tricarboxylic acid cycle); and other attributes contributing to
their ecological and evolutionary significance (2).
Chlorobium tepidum is a moderately thermophilic green sulfur
bacterium. It grows rapidly on a defined medium and makes a suitable model for genetic, biochemical, and physiological studies of the green
sulfur bacteria (3, 22). The 2.15-Mb genome of C. tepidum has recently been sequenced and reveals about 2,284 open
reading frames of which about 50% have been assigned a known function (J. A. Eisen et al., unpublished data).
A powerful way to investigate the function of the genes of an organism
is by targeted gene inactivation by homologous recombination (20). The success of such an approach typically depends on
the availability of an antibiotic which effectively inhibits the growth of wild-type cells and to which spontaneously resistant mutants are not
easily formed, on a selection marker which confers resistance to this
antibiotic, and on a method to introduce DNA into the cells.
Transformation of Chlorobium species using natural
transformation (5, 12), chemical transformation
(10), and electroporation (9) has previously
been reported. Spectinomycin and streptomycin were the only antibiotics
used for selection in these transformations (5, 9, 12).
Ampicillin and chloramphenicol resistance markers have also been
reported to be useful for selection in conjugation studies of C. tepidum (24). At present, the only genes in any
Chlorobium species that have been reported as targets for
inactivation encode chlorosomal proteins CsmC and CsmA
(5), reaction center cytochrome
c551 PscC (9), and
Rubisco subunit RbcL (T. E. Hanson and F. R. Tabita, personal
communication). Of these, only the csmC (5) and
rbcL mutants (T. E. Hanson and F. R. Tabita,
personal communication) fully segregated.
C. tepidum is a nitrogen-fixing organism (23),
and in the present study the nifD gene, which encodes a
subunit of nitrogenase (15), was studied as a general
model for gene inactivation. This allowed facile detection of true
transformants as mutants that had a known phenotype, namely, the
inability to grow diazotrophically. Several antibiotics and resistance
markers were tested, and three markers were found to be suitable for
use in C. tepidum: a spectinomycin-streptomycin resistance
marker, a gentamicin resistance marker, and an erythromycin resistance
marker. Natural transformation of C. tepidum was also characterized as a general method for gene inactivation by homologous recombination.
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MATERIALS AND METHODS |
Organisms and growth conditions.
The strain of C. tepidum used for transformation was WT2321 (24),
which is a plating strain derived from C. tepidum strain ATCC 49652 (22). Growth conditions were essentially as
previously described (24). All growth and manipulations of
C. tepidum were performed in an anaerobic chamber (Coy
Laboratory Products, Grass Lake, Mich.) which had an atmosphere of 10%
CO2 and 5% H2 balanced with N2. C. tepidum was grown at
40°C throughout the study.
All cloning was carried out with Escherichia coli DH5
grown in Luria-Bertani medium. Antibiotic selection conditions
for E. coli were obtained with 100 µg of ampicillin
ml1 combined with 10 µg of gentamicin, 100 µg of spectinomycin, or 10 µg of chloramphenicol
ml1. Ampicillin, gentamicin, and kanamycin were
obtained from U.S. Biochemicals (Cleveland, Ohio), and other
antibiotics were obtained from Sigma (St. Louis, Mo).
One liter of liquid medium for C. tepidum (CL) was made of
20 ml of salts A (0.64 g of Na2 · EDTA · 2H2O, 10 g of
MgSO4 · 7H2O,
2.5 g of CaCl2 · 2H2O, and 20 g of NaCl per liter), 20 ml of
salts B (25 g of NH4CH3COO,
20 g of NH4Cl, and 115 g of
Na2S2O3 · 5H2O per liter), 20 ml of buffers (25 g of
KH2PO4 and 105 g of
MOPS [3-{N-morpholino}propanesulfonic acid] per
liter), 1 ml of trace elements (22), 50 µl of 10-mg
ml1 resazurin, and 20 µl of 1-mg
ml1 vitamin B12. After
the medium was autoclaved at 121°C for 20 min, a freshly made,
filter-sterilized solution of 0.6 g of
Na2S · 9H2O and
2.0 g NaHCO3 in 50 ml of water was added.
The pH of the medium was usually within the desired range of 6.9 to 7.0 without further treatment but otherwise was aseptically adjusted with 1 M NaOH or 1 M HCl.
One liter of plating medium for C. tepidum (CP) made about
25 plates and contained 20 ml of salts A, 20 ml of salts B, 20 ml of
buffers, 1 ml of trace elements, 50 µl of 10-mg
ml1 resazurin, 20 µl of 1-mg
ml1 vitamin B12, and
0.36 g of L-cysteine. The pH was adjusted to 7.6 with 10 M NaOH, and 15 g of agar (Bacto Agar; Becton
Dickinson, Sparks, Md.; used as supplied) per liter was added before
autoclaving. The medium was cooled to 50°C in a water bath before
addition of antibiotics and the pouring of the plates. The plates were poured in a cold room to allow rapid solidification and moved to the
anaerobic chamber in less than 20 min to prevent excessive oxidation of
the cysteine. The final pH of the plates was approximately 6.8 to 7.0. Ammonium-free plating medium (CPNF) for diazotrophic growth was made by
substituting salts BNF (26.5 g of NaCH3COO and
115 g of
Na2S2O3
· 5H2O per liter) for salts B and leaving out
cysteine. Once inoculated, the plates were placed in an anaerobic jar
(BBL GasPak 100 system; Becton Dickinson) without palladium catalyst
but containing one disposable
H2-CO2-generating envelope (BBL GasPak; Becton Dickinson) and a small tube with approximately 0.1 g of thioacetamide; H2S generation from
the thioacetamide was activated by adding approximately 1 ml of 1 M
HCl. The jars were kept in dim light for 1 to 2 h before transfer
to the appropriate temperature and illumination conditions. In addition
to ambient illumination, a single 100-W incandescent light bulb was
placed 0.4 to 0.8 m from the jars. The jars were always kept
inside the anaerobic chamber.
Transformation of C. tepidum.
Unless
otherwise stated, the standard protocol for agar plate transformation
was as follows. Cells from 100 µl of an overnight culture of C. tepidum in the late exponential growth phase (approximately 3 × 109 to 6 × 109
cells ml1) were harvested in a microcentrifuge
tube, resuspended in 20 µl of CL medium containing 1 µg of DNA, and
spotted on a nonselective CP plate in an area with a diameter of 6 to 8 mm. The plate was placed in a jar and kept in the dark for 1 to 2 h prior to incubation in the light at 40°C for 18 to 20 h. The
cell patch was then scraped off and suspended in 300 µl of CL. This
suspension and dilutions thereof were spread on selective and
nonselective CP plates and incubated for 5 to 6 days to allow single
colonies to appear. The mutation frequency was calculated as the number
of antibiotic-resistant mutants counted on the selective plates divided
by the total number of viable cells counted on the nonselective plates.
All transformation frequencies represented in the figures and tables
represent the means of up to four separate experiments in which the
standard deviations were less than 50% of the means.
Preparation of plasmid and genomic DNA.
Plasmids were
prepared by alkaline lysis (19). Digested plasmids to be
used for transformation were extracted with an equal volume of
chloroform-isoamyl alcohol (24:1 by volume), precipitated with 0.1 volume of 3 M ammonium acetate and 1 volume of isopropanol at 
20°C,
washed with 70% (vol/vol) ethanol, and redissolved in sterile water.
Genomic DNA from C. tepidum and Synechococcus sp.
strain PCC 7002 was prepared using an unpublished method developed by
Dexter Chisholm (DuPont, Wilmington, Del.). Cells from 5 to 20 ml of culture were harvested and incubated in 500 µl of TES buffer (5 mM
Tris, 5 mM EDTA, 50 mM NaCl, pH 8.5) containing 10 mg of lysozyme ml1 for 0.5 to 1 h at 37°C. Sodium
Sarkosyl (50 µl of a 10% [wt/vol] stock solution) was added, and
the suspension was extracted twice with 600 µl of buffered phenol.
RNase was added to a final concentration of 100 µg
ml1, and the mixture was incubated for 30 min
at 37°C. To this solution, NaCl (100 µl of a 5 M stock solution)
and hexadecyltrimethylammonium bromide (CTAB; 100 µl of a stock
solution of 10% [wt/vol] CTAB-0.7 M NaCl) were added; the mixture
was then extracted twice with 600 µl of chloroform-isoamyl alcohol
(24:1 by volume). DNA in the aqueous phase was precipitated with 600 µl of isopropanol, washed with 70% (vol/vol) ethanol, and
redissolved in sterile water. Genomic DNA for PCR analysis was isolated
from cells grown on plates. Genomic DNA for Southern hybridization
analysis was isolated from cells grown to the late exponential phase in
liquid medium because it appeared to be difficult to completely digest DNA isolated from cells grown on plates.
PCR conditions.
Oligonucleotide primers were designed based
on the genomic sequence (J. A. Eisen et al., unpublished data)
using MacVector software, version 6.5 (Genetics Computer Group,
Madison, Wis.) and synthesized at the Nucleic Acid Facility, The
Pennsylvania State University. The PCR conditions were as follows:
initially 5 min at 95°C, and then 35 cycles of 1 min at 95°C, 1 min
at 58°C, and 2 min at 72°C for PCR products <2 kb, and ultimately
10 min at 72°C. For PCR products of 2 to 3 kb, the elongation time at 72°C was increased to 3 min.
Construction of plasmids.
Figure
1a shows a map of a portion of the
nif region of C. tepidum based on the complete
genomic sequence (J. A. Eisen et al., unpublished data). A 2.93-kb
fragment was amplified from genomic DNA using primers nif-F2,
5'-GGAATTCGCGTCGGCGATGTGGTCTAT, and nif-B2,
5'-GGAATTCGTCGGAGGTGTCTGGGAA.
(In the primer sequences, heterologous bases are italicized and
EcoRI recognition sites are underlined.) Plasmid pTN1 was
produced by digesting this PCR product with EcoRI and
cloning the product into the EcoRI site of pUC19 (Table
1). Plasmid pTN1G4 was made by inserting the aacC1 cassette from pMS266 (Table 1 and Fig. 1c) into
the XhoI site of nifD in pTN1 (Fig.
2a). Similar plasmid constructs with
nifD::aacC1 but with shorter flanking
regions of C. tepidum DNA were also made: pTN2G1 (primers
nif-F3,
5'-GGAATTCAGGGCGTGGTTCTTGGTCC, and nif-B3,
5'-GGAATTCGAGTTCGGCTTTGCTCTTT)
and pTN3G11 (primers nif-F5L,
5'-GGGAATTCGCTGGTCACCACATCGCAA,
and nif-B5L,
5'-CCATGGAATTCCGTACTTGGTCTC)
were made by PCR with pTN1G4 and by cloning the PCR products into the
EcoRI site of pUC19 (Fig. 2a and Table 1). pTN1G4, pTN2G1,
and pTN3G11 linearized with AhdI were used for
transformation and are depicted in Fig. 2b to d. pTN1S3 was made by
inserting the aadA cassette from pHP45
(Fig. 1b) into the
XhoI site of pTN1, and pTN1CE1 was made by inserting the
ermC-cat cassette from pRL409 (Fig. 1d) into the XhoI site of pTN1 (Table 1).
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FIG. 1.
(a) Map of part of the nif region in
C. tepidum; (b) map of the aadA
streptomycin-spectinomycin resistance cassette from pHP45; (c) map
of the aacC1 gentamicin resistance cassette from pMS266;
(d) map of the ermC-cat erythromycin and chloramphenicol
resistance cassette from pRL409. The positions of some of the primers
discussed in the text are also shown.
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FIG. 2.
(a) Map of plasmid pTN1G4 also showing the positions of
some of the primers discussed in the text; (b)
AhdI-digested pTN1G4; (c) AhdI-digested
pTN2G1; (d) AhdI-digested pTN3G11. Line, pUC19
DNA; light grey, C. tepidum DNA; dark grey, gentamicin
resistance marker.
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Genomic DNA analysis of C. tepidum
transformants.
Detection of wild-type nifD and
cassette-interrupted nifD was performed by PCR with primers
nif-F4, 5'-CACCACATCGCAAACAAC, and nif-B4
5'-GCAGGAACCTCTTCGGCAATC (Fig. 1a). PCR detection of the
aacC1 cassette was performed with primers GmR-F1,
5'-GTGACGCACACCGTGGAAAC, and GmR-B1,
5'-TCCCGTATGCCCAACTTTGTA (Fig. 1c). Southern hybridization was carried out as described previously (19) with
[-32P]dATP-labeled probes (Random Primed DNA
labeling kit; Boehringer Mannheim, Indianapolis, Ind.). Hybridizing DNA
fragments were detected with a PhosphorImager 445-SI (Molecular
Dynamics, Sunnyvale, Calif.). A 413-bp probe for nifD was
made by PCR with primers nif-F4 and nif-B4 (Fig. 1a). A 559-bp probe
for the ermC-cat cassette was made by PCR with primers
CmR-F1, 5'-ACGGGGGCGAAGAAGTTGTC and CmR-B1,
5'-CGGGCGTATTTTTTGAGTTATCG (Fig. 1d). A 1.1-kb probe for aadA was excised from plasmid pSRA2, which contains an
aadA cassette derived from pHP45 (N.-U. Frigaard and
D. A. Bryant, unpublished data).
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RESULTS AND DISCUSSION |
Antibiotic sensitivity.
Because the antibiotic resistance
markers used in this study originate from mesophiles, the temperature
used in the transformation experiments was lower than the optimum 47 to
48°C growth temperature of C. tepidum. To test the
antibiotic sensitivity of wild-type C. tepidum, cells from
100 µl of a late-exponential culture were plated on CP plates with
increasing concentrations of antibiotics and incubated at 40°C. The
following concentrations were found to inhibit growth completely, and
these concentrations were used throughout the present study:
gentamicin, 100 µg ml1; erythromycin, 2 µg
ml1; chloramphenicol, 30 µg
ml1; tetracycline, 1 µg
ml1. Streptomycin and spectinomycin only
efficiently inhibited growth when combined. Therefore, streptomycin
(300 µg ml1) and spectinomycin (150 µg
ml1) were used in combination, since the
aadA cassette from pHP45 confers resistance to both
antibiotics. Spontaneous mutants resistant to gentamicin, erythromycin,
chloramphenicol, tetracycline, or streptomycin-spectinomycin were not
observed when the wild-type culture was plated on plates with the
stated concentration of antibiotic. Kanamycin and ampicillin did not
completely inhibit growth on plates at concentrations below 100 µg
ml1.
Previous work (11) with other Chlorobium
species suggests that amoxicillin, nalidixic acid, vancomycin,
mitomycin C, and colistin might also efficiently inhibit C. tepidum. However, these antibiotics were not tested in this study.
Optimization of transformation.
Figure
3 shows the number of transformants and
the transformation frequency when C. tepidum cells from a
100-µl late-exponential-phase culture were incubated with 1 µg of
AhdI-digested pTN1G4 on a CP plate for various times. The
transformation frequency reached about 2 × 107 to 3 × 107 at
10 h and increased only slightly thereafter. (This corresponds to
a yield of approximately 102 transformants per
µg of DNA at 10 h.) This suggests that most of the
transformation events occurred at the beginning of the experiment
(before 10 h) and that the transformation events were stable.
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FIG. 3.
Transformation of C. tepidum incubated
for various periods. Solid circles, numbers of gentamicin-resistant
transformants; open circles, transformation frequencies. See text for
details.
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Transformation was also attempted in a liquid suspension with the same
amount of cells and DNA as described above for transformation on an
agar plate. The cells were washed and incubated in 100 µl of fresh CL
medium containing 1 µg of DNA for various periods between 1 and
25 h and then plated on selective CP plates. The highest
transformation frequency obtained was about an order of magnitude lower
than the transformation frequencies obtained on agar plates (data not
shown). When the cells and DNA are spotted on a solid agar surface and
allowed to dry, the cells and DNA may interact differently than in a
liquid suspension. This may allow an increased uptake of DNA by the
cells and thus increase the transformation frequency.
Previous work with Chlorobium limicola strain 8327 showed
that these cells are competent in both the exponential and stationary growth phases (12). The exact growth state of the cells
used for transformation was also not critical in our transformation protocol for C. tepidum. When equal volumes of an overnight
culture in the late exponential growth phase and a 4-day-old
culture in the stationary growth phase were used in a 20-h
transformation, the stationary cells gave about one-half as many
transformants as the late-exponential-phase cells (data not shown). In
both cases, the spotted cells exhibited visible growth and sulfur
formation during the 20-h incubation on nonselective plates.
Transformation with increasing amounts of linearized DNA resulted in an
increased transformation frequency (Table
2). Increasing the DNA amount 100-fold
from 0.1 to 10 µg only increased the transformation frequency about
3-fold; this suggests that 10 µg of DNA is close to a saturating
amount of DNA for the number of cells used. The transformation
frequency with 1 µg of linearized plasmid (EcoRI- or
AhdI-digested pTN1G4) was about an order of magnitude higher than that for the same amount of circular plasmid (undigested pTN1G4)
(data not shown). The reason for this difference is not clear but
probably results from the DNA binding and uptake mechanisms of the
cells. These observations suggest that at least 1 µg of linearized
plasmid is suitable for routine transformation of C. tepidum.
Effect of variation in length of homologous flanking DNA.
When
a plasmid construct is made for gene inactivation by homologous
recombination, it is usually advantageous to include a large region of
homologous DNA to increase the probability of homologous recombination.
However, restriction endonuclease sites and toxic gene products may
impose practical restrictions on the length of homologous DNA that can
easily be cloned. To determine the effect of the length of homologous
flanking DNA on transformation of C. tepidum, three
constructs for nifD inactivation were made; these contained
a total of 2.93, 1.08, and 0.29 kb of flanking homologous DNA in which
the aacC1 gentamicin resistance marker was inserted
approximately in the middle (Table 1 and Fig. 2). These constructs were
digested with either AhdI, which cuts the plasmids only once
and which leaves flanks of pUC19 DNA (Fig. 2), or with
EcoRI, which cuts twice and which excises all of the pUC19
DNA (Fig. 2a). The transformation frequencies with 2.93 kb of
homologous DNA were similar regardless of whether the plasmid was
digested with EcoRI or AhdI (Table
3). But with 1.08 kb of homologous DNA,
the transformation frequency was an order of magnitude lower when the
plasmid was digested with EcoRI than when the plasmid was
digested with AhdI. No transformation was observed with only 0.29 kb of homologous DNA regardless of the enzyme used for
linearization. Some bacteria partially degrade absorbed DNA via
exonuclease activity (14), and this may be the case for
C. tepidum as well. Therefore, it may be advantageous to
include dispensable DNA at the ends of the linearized DNA used for
transformation. Such exonuclease activity might explain the difference
in transformation frequency with pTN2G11 depending on the enzyme used
for linearization (Table 3).
These observations suggest that a region of homologous flanking DNA of
about 1 kb should be suitable for routine transformation experiments
and that it may be advantageous to linearize plasmids with a
restriction enzyme that leaves dispensable flanking DNA at the ends of
the fragment. Separate fragments of nontransforming DNA produced by an
enzyme digest (e.g., the pUC19 vector residue excised by
EcoRI from pTN1G4) may be disadvantageous because this nontransforming DNA may compete with the transforming DNA for uptake
into the cells. Inhibition of transformation by competing DNA was
demonstrated by another observation. Addition of 20 µg of sonicated
chromosomal DNA from Synechococcus to a transformation mixture of C. tepidum containing 1 µg of linearized DNA
(AhdI-digested pTN1G4) decreased the transformation
frequency an order of magnitude (data not shown).
Various selection markers.
Three constructs for
nifD inactivation were made with different antibiotic
resistance markers, pTN1G4, pTN1S3, and pTN1CE1 (Table 1). The
transformation frequencies were about the same when the
aacC1 gentamicin resistance marker and the
ermC-cat erythromycin-chloramphenicol resistance marker were
used (Table 4.) Antibiotic-resistant
mutants were only obtained with the ermC-cat marker when
erythromycin was used as the selective agent and not when
chloramphenicol was used. The obtained Emr
mutants were not Cmr even though the marker
contains both the cat and ermC genes (Fig. 1d).
Southern hybridization analysis confirmed that the cat
marker was present in the Emr mutants (see below;
Fig. 4). Thus, this cat marker
did not function in C. tepidum, probably either because the
expressed Cat protein is not functional in C. tepidum or
because the cat promoter is too weak in C. tepidum. Our failure with the cat marker is in contrast
to the conjugation studies by Wahlund and Madigan (24), who successfully used a cat marker similar to ours for
selection in C. tepidum. Previous work with the tetracycline
resistance marker (tet) from pBR325 suggested that this
marker does not work in C. tepidum (24), and
this marker was not investigated further in this study. It is possible
that both the tet and cat markers may work in
C. tepidum if their promoters are replaced, e.g., with the
promoter from ermC in pRL409 or from aacC1 in
pMS266 or with a strong indigenous promoter such as that for
csmCA (4, 5, 8).
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FIG. 4.
Growth of wild-type C. tepidum and
antibiotic-resistant transformants spotted on nonselective CP plates
(a, c, and e) and on nonselective CPNF plates (b, d, and f). (a and b)
22 Emr transformants; (c and d) 18 Gmr
transformants; (e and f) 22 Smr/Spr
transformants. As controls, wild-type C. tepidum
(labeled 2) and a confirmed Nif transformant (labeled 1)
were also spotted on all plates.
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A transformation frequency 4 orders of magnitude higher than that
obtained with the aacC1 and ermC markers was
observed with the aadA marker (Table 4). Analysis of the
obtained mutants resistant to the combination of streptomycin and
spectinomycin (Smr/Spr
mutants) showed that all of 22 mutants analyzed were incapable of diazotrophic growth and that nifD therefore was
inactivated (see below). The reason for this increased transformation
frequency is not clear, but the genomic sequence of C. tepidum may hold some clues. In contrast to the aacC1
and ermC-cat markers, the aadA marker from
pHP45 contains a 59-bp recombinational hot spot immediately
downstream of aadA (17). This hot spot is
recognized by IntI-like integrases (6, 18), and the
C. tepidum genome contains an integrase (CT0176) with high
homology to this class of enzymes (J. A. Eisen et al., unpublished
data). The hot spot in the aadA marker and the indigenous
integrase CT0176 in C. tepidum may be related to the high
transformation frequency of C. tepidum with
aadA-containing plasmid pTN1S3. However, regions of C. tepidum DNA were necessary to obtain high transformation
frequencies with the aadA marker. Transformation with a
linear DNA fragment encoding only the aadA marker
(PstI-digested pHP45) resulted in a transformation frequency of only approximately 3 × 108,
which is 5 orders of magnitude lower than the transformation frequency
obtained with linearized pTN1S3 (Table 4). The C. tepidum genome also contains a gene (encoding CT1017) with a downstream 59-bp recombinational hot spot (J. A. Eisen et al., unpublished data). CT1017 has no significant sequence similarity with any protein
in GenBank, but the gene seems to encode a cytoplasmic protein and
could be a novel antibiotic resistance-encoding gene (18).
Test of transformants.
The expected phenotype of the C. tepidum nifD transformants is the inability to reduce dinitrogen.
Several mutants obtained by transformation with the three different
markers (Table 4) were transferred to selective CP plates three times
and then transferred to nonselective CP and nonselective CPNF plates to
check for diazotrophic growth (Fig. 4). Wild-type C. tepidum
grew on CPNF plates although slightly slower than on CP plates. All
Gmr mutants (23 tested), all
Smr/Spr mutants (22 tested), and nearly all Emr mutants (23 out of 24 tested) failed to grow on the CPNF plates. These results confirm that
the mutants had lost the ability to perform nitrogen fixation and that
the mutations had segregated completely.
Genomic DNA was isolated from five Gmr mutants
and analyzed by PCR. The results from one of the mutants are shown in
Fig. 5a. As expected, PCR with primers
specific for nifD (nif-F4 and nif-B4) amplified a 0.41-kb
fragment in the wild type and a 1.46-kb fragment in the mutants. PCR
with primers specific for aacC1 (GmR-F1 and GmR-B1) did not
produce a PCR product in the wild type but amplified a 0.75-kb fragment
in the mutants.
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FIG. 5.
(a) PCR analysis of wild-type C. tepidum
(lanes 1 and 3) and a Gmr mutant (lanes 2 and 4). Primers
specific for nifD, nif-F4 and nif-B4, were used in lanes
1 and 2, and primers specific for the gentamicin resistance cassette
(accC1), GmR-F1 and GmR-B1, were used in lanes 3 and 4. (b) Southern hybridization analysis of wild-type C.
tepidum (lanes 1 and 3) and an Emr mutant (lanes 2 and 4). A probe specific for nifD was used in lanes 1 and 2, and a probe specific for the erythromycin-chloramphenicol
resistance cassette (ermC-cat) was used in lanes 3 and
4.
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Genomic DNA was also isolated from three Emr
Nif mutants and analyzed by PCR and Southern
hybridization. As expected, PCR with primers nif-F4 and nif-B4 did not
produce a 0.41-kb fragment in the mutants. PCR with primers specific
for cat (CmR-F1 and CmR-B1) did not produce a PCR product in
the wild type but amplified a 0.56-kb fragment in the mutants. PCR with
primers CmR-B1 and nif-B4 did not produce a PCR product in the wild
type but amplified a 1.3-kb fragment in the mutants, which confirms the
insertion of the ermC-cat marker in nifD in the
expected orientation. Figure 5b shows a Southern hybridization analysis
of StuI-digested genomic DNA from an
Emr Nif mutant. As
expected, a nifD probe hybridized with a 0.9-kb fragment in
the wild type and with a 3.6-kb fragment in the mutant. A
cat probe did not hybridize with wild-type genomic DNA but
hybridized with a 3.6-kb fragment in the mutant. PCR analysis of
genomic DNA isolated from the only Emr mutant
that exhibited diazotrophic growth showed the presence of wild-type
nifD using primers nif-F4 and nif-B4 and the absence of
cat using primers CmR-F1 and CmR-B1. This
Emr Nif+ mutant could
therefore represent a spontaneously resistant mutant or an
Emr transformant produced by an illegitimate
recombination event.
Six Smr/Spr transformants
were likewise analyzed by digesting genomic DNA with StuI
and performing a Southern hybridization analysis (data not shown). As
expected, the nifD probe hybridized with a 0.9-kb fragment
in the wild type and with a 2.9-kb fragment in the mutants. The
aadA probe did not hybridize with wild-type genomic DNA but
hybridized with a 2.9-kb fragment in all six transformants.
Conclusion.
Genes in C. tepidum can be
insertionally inactivated by natural transformation and homologous
recombination. Markers for resistance to gentamicin (aacC1
from pMS266), erythromycin (ermC from pRL409), and
streptomycin-spectinomycin (aadA from pHP45) were
successfully used in the present study to inactivate nifD.
The aadA marker gave a significantly higher transformation
yield than the two other markers. We suggest the following general
guidelines for routine gene inactivation by natural transformation: (i)
cells from at least 100 µl of a late-exponential liquid culture
should be used; (ii) linearized DNA (1 to 10 µg) with sequences of at least 0.5 kb of homologous DNA flanking each side of the selection marker should be used; (iii) transforming cells should be spotted on an
agar surface and incubated for 10 to 20 h at 40°C; shorter incubation times can probably be used, especially if the incubation temperature is higher. Finally, in its simplest form, transformation may be performed by scraping cells off a plate and incubating a mixture
of these cells and transforming DNA on a nonselective plate overnight.
The cells should then be restreaked on selective plates the next day.
| |
ACKNOWLEDGMENTS |
N.-U.F. was supported by The Danish Natural Science
Research Council. This work was supported by U.S. Department of Energy grant DE-FG02-97ER20137 to D.A.B.
We thank the Institute of Genomic Research for prepublication access to
the genome sequence of C. tepidum.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, 232 S. Frear Building, The
Pennsylvania State University, University Park, PA 16802. Phone: (814)
863-7405. Fax: (814) 863-7024. E-mail: nxf10{at}psu.edu.
| |
REFERENCES |
| 1.
|
Becker, A.,
M. Schmidt,
W. Jäger, and A. Pühler.
1995.
New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions.
Gene
162:37-39[CrossRef][Medline].
|
| 2.
|
Blankenship, R. E.,
M. T. Madigan, and C. E. Bauer.
1995.
Anoxygenic photosynthetic bacteria.
Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 3.
|
Castenholz, R. W.,
J. Bauld, and B. B. Jørgensen.
1990.
Anoxygenic microbial mats of hot springs: thermophilic Chlorobium sp.
FEMS Microbiol. Ecol.
74:325-336.
|
| 4.
|
Chung, S.,
G. Frank,
H. Zuber, and D. A. Bryant.
1994.
Genes encoding two chlorosome components from the green sulfur bacteria Chlorobium vibrioforme strain 8327D and Chlorobium tepidum.
Photosynth. Res.
41:261-275[CrossRef].
|
| 5.
|
Chung, S.,
G. Shen,
J. Ormerod, and D. A. Bryant.
1998.
Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable.
FEMS Microbiol. Lett.
164:353-361[CrossRef][Medline].
|
| 6.
|
Collis, C. M.,
M.-J. Kim,
H. W. Stokes, and R. M. Hall.
1998.
Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites.
Mol. Microbiol.
29:477-490[CrossRef][Medline].
|
| 7.
|
Elhai, J., and C. P. Wolk.
1988.
A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.
Gene
68:119-138[CrossRef][Medline].
|
| 8.
|
Gruber, T. M., and D. A. Bryant.
1998.
Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green nonsulfur bacterium Chloroflexus aurantiacus.
Arch. Microbiol.
170:285-296[CrossRef][Medline].
|
| 9.
|
Kjærulff, S.,
D. B. Diep,
J. S. Okkels,
H. V. Scheller, and J. G. Ormerod.
1994.
Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium Chlorobium vibrioforme by homologous recombination.
Photosynth. Res.
41:277-283[CrossRef].
|
| 10.
|
Méndez-Alvarez, S.,
V. Pavón,
I. Esteve,
R. Guerrero, and N. Gaju.
1994.
Transformation of Chlorobium limicola by a plasmid that confers the ability to utilize thiosulfate.
J. Bacteriol.
176:7395-7397[Abstract/Free Full Text].
|
| 11.
|
Nogales, B.,
R. Guerrero, and I. Esteve.
1994.
Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents.
FEMS Microbiol. Lett.
123:37-42[CrossRef][Medline].
|
| 12.
|
Ormerod, J. G.
1988.
Natural genetic transformation in Chlorobium, p. 315-319.
In
J. M. Olson, J. G. Ormerod, J. Amesz, E. Stackebrandt, and H. G. Trüper (ed.), Green photosynthetic bacteria. Plenum Press, New York, N.Y.
|
| 13.
|
Overmann, J., and C. Tuschak.
1997.
Phylogeny and molecular fingerprinting of green sulfur bacteria.
Arch. Microbiol.
167:302-309[CrossRef][Medline].
|
| 14.
|
Palmen, R., and K. J. Hellingwerf.
1997.
Uptake and processing of DNA by Acinetobacter calcoaceticus a review.
Gene
192:179-190[CrossRef][Medline].
|
| 15.
|
Postgate, J.
1998.
Nitrogen fixation, 3rd ed.
Cambridge University Press, Cambridge, United Kingdom.
|
| 16.
|
Prentki, P., and H. M. Krisch.
1984.
In vitro insertional mutagenesis with a selectable DNA fragment.
Gene
29:303-313[CrossRef][Medline].
|
| 17.
|
Prentki, P.,
A. Binda, and A. Epstein.
1991.
Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon.
Gene
103:17-23[CrossRef][Medline].
|
| 18.
|
Recchia, G. D., and R. M. Hall.
1995.
Gene cassettes: a new class of mobile element.
Microbiology
141:3015-3027[Medline].
|
| 19.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 20.
|
Thiel, T.
1994.
Genetic analysis of cyanobacteria, p. 581-611.
In
D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 21.
|
Trüper, H. G., and N. Pfennig.
1992.
The family Chlorobiaceae, p. 3583-3592.
In
A. Balows, H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer-Verlag, New York, N.Y.
|
| 22.
|
Wahlund, T. M.,
C. R. Woese,
R. W. Castenholz, and M. T. Madigan.
1991.
A thermophilic green sulfur bacterium from New Zealand hot springs, Chlorobium tepidum sp. nov.
Arch. Microbiol.
156:81-90[CrossRef].
|
| 23.
|
Wahlund, T. M., and M. T. Madigan.
1993.
Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum.
J. Bacteriol.
175:474-478[Abstract/Free Full Text].
|
| 24.
|
Wahlund, T. M., and M. T. Madigan.
1995.
Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum.
J. Bacteriol.
177:2583-2588[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, June 2001, p. 2538-2544, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2538-2544.2001
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Abstract
Introduction
