Department of Biochemistry and Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas, 18008 Granada, Spain
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INTRODUCTION |
Pseudomonas putida is a
member of rRNA group I of the genus Pseudomonas. This
species is able to colonize many different niches, including soil,
freshwater, and the surfaces of living organisms (e.g., the roots of
agriculturally important plants) (9, 37, 42, 49, 60). A
number of different strains have been isolated from these niches, and a
relevant property of all of them is the ability to metabolize a wide
range of biogenic and xenobiotic compounds. P. putida mt-2
was isolated from soils by virtue of its ability to use
3-methylbenzoate as the sole C source (41), a property later
shown to be associated with the presence of the TOL plasmid pWW0
(76). P. putida KT2440 is a cured,
restriction-deficient derivative of P. putida mt-2
(13) which has been widely used in physiological and genetic
studies (see references 48 and 51
for reviews). The nonpathogenic P. putida KT2440 has been shown to be an ideal host for expanding the range of substrates that it
can degrade through the recruitment of genes from other microorganisms
(52, 53). This strain has also been used as a vehicle for
gene cloning and expression (36) and for the
biotransformation of several chemicals in added-value products
(8). This strain also colonizes the plant rhizosphere, which
makes it potentially useful for phytorhizoremediation and for the
development of biopesticides. These features make P. putida
KT2440 a key strain within this genus (37, 49, 60). An
international consortium is now considering sequencing its genome
(73).
For efficient exploitation of this strain, thorough knowledge of its
genome organization is essential. The development of pulsed-field gel
electrophoresis (PFGE) and concomitant technology for the manipulation
of large fragments of DNA (68) have revolutionized the
analysis of bacterial chromosomes. More than 100 physical maps have
been constructed (5, 12). For three members of the genus
Pseudomonas
Pseudomonas aeruginosa PAO1,
Pseudomonas fluorescens SBW25, and Pseudomonas
syringae pv. phaseolicolacomplete physical maps and detailed
genetic maps have been constructed (7, 19, 46, 57, 65). In
the case of P. aeruginosa, a sequencing project is providing
in-depth knowledge about the species.
Unlike the situation for P. aeruginosa, little is known
about the P. putida genome, and only an approximate
chromosome map for one of the strains of this species has been
generated, by conjugation and transduction analysis (38,
71). We present here a macrorestriction map of P. putida KT2440 developed from data obtained by PFGE and Southern
blot analyses. The size of the circular chromosome was estimated to be
6.0 Mb, based on the size of the fragments generated from the digestion
of the whole chromosome with I-CeuI, PacI,
SwaI, and PmeI.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and culture conditions.
P.
putida EEZ15, a phosphinothricin-resistant derivative of the
prototroph P. putida KT2440, was described in an earlier
publication (56). P. putida EEZ15K-1 through -34 are kanamycin-resistant (Kmr) mini-Tn5
derivatives of P. putida EEZ15 (56).
Escherichia coli JM109 was used to maintain different
plasmids (66). Bacterial cells were grown at 30°C on
Luria-Bertani (LB) culture medium. When necessary, ampicillin,
chloramphenicol, kanamycin, and tetracycline were added to final
concentrations of 100, 180, 25, and 15 µg/ml, respectively. Plasmids
used for preparation of gene probes were extracted from E. coli host strains by the alkaline lysis method (66).
PFGE. (i) DNA preparation.
Unsheared DNA was prepared by
embedding whole cells in agarose blocks. P. putida cells
were grown in LB to late exponential phase; to align the origins of
chromosomal replication, the culture was supplemented with
chloramphenicol (180 µg/ml) and maintained for another hour. Cells
were harvested by centrifugation for 10 min at 1,400 × g and washed twice with PettIV buffer (10 mM Tris-HCl [pH
7.6], 1 M NaCl) (1). Cell suspensions of different cell densities were prepared in the same buffer and mixed with an equal volume of molten 1.6% (wt/vol) low-melting-point preparative-grade agarose (Bio-Rad) to obtain agarose plugs with 0.5 × 109, 1 × 109, and 2 × 109 cells/ml. Cells were lysed by submerging the plugs in
EC-lysis solution (6 mM Tris-HCl [pH 7.6], 1 M NaCl, 100 mM EDTA [pH
7.5], 0.5% [wt/vol] Brij 58, 0.2% [wt/vol] deoxycholate, 0.5%
[wt/vol] sarcosyl, 1 mg of hen egg white lysozyme per ml, 20 µg of
bovine pancreatic RNase per ml) for 24 h at 37°C. The EC-lysis
solution was replaced with ESP solution (0.5 M EDTA [pH 9.5], 1%
[wt/vol] lauryl sarcosine, 1 mg of proteinase K per ml), and
incubation continued for a further 48 h at 50°C. The agarose
blocks were washed several times with TE buffer (10 mM Tris-HCl, 10 mM
EDTA [pH 8]) and stored at 4°C until use.
(ii) Restriction endonuclease digestion and end labeling.
Endonucleases were purchased from New England Biolabs (PmeI,
PacI, and I-CeuI) and Boehringer Mannheim
(SwaI). Before restriction, one-third of an agarose block
was equilibrated three times with 1 ml of the recommended restriction
buffer, replaced with 60 µl of fresh restriction buffer supplemented
with 20 µg of bovine serum albumin per ml, 7 mM dithiothreitol, and
the appropriate amount of enzyme (4, 1, 2, and 5 U for PmeI,
I-CeuI, PacI, and SwaI, respectively),
incubated overnight at 4°C, and then incubated at 37°C for 2 h. Double digestion was done sequentially. End labeling of
PmeI- or SwaI-digested DNA was achieved by
incubating each plug with 5 µCi of [
-32P]dTTP in 20 µl of Klenow buffer (10 mM MgCl2, 50 mM NaCl, 10 mM
Tris-HCl [pH 7.5]) with 1 U of Klenow enzyme for 30 min at room
temperature. 32P-labeled fragments were separated by
conventional gel electrophoresis and transferred to nylon membranes as
described below. 32P-labeled fragments were detected by autoradiography.
(iii) CHEF electrophoresis.
Contour-clamped homogeneous
electric field (CHEF) electrophoresis was done in a Pharmacia-LKB Gene
Navigator. Agarose gels (1.2% [wt/vol]) were run in 0.5× TBE buffer
(45 mM Tris-borate, 45 mM boric acid, 1 mM EDTA [pH 8]) at 10°C
unless otherwise stated. Voltage, pulse time, and total running time
varied according to the size range of fragments to be separated.
Specific conditions are provided in the legends for the corresponding
figures. Lambda DNA concatemers (Pharmacia), laboratory-made lambda
HindIII digest fragments, and chromosomes of
Saccharomyces cerevisiae S-13 and Hansenula
wingei (Bio-Rad) were used as molecular size DNA standards.
Southern hybridization and DNA labeling.
DNA fragments
separated in PFGE gels were irradiated with UV light (254 nm) for 2 min. DNA was transferred onto nylon membranes by capillary blotting for
48 h (70). Specific probes for hybridization were
recovered from agarose gels with an agarose gel DNA extraction kit
(Boehringer Mannheim). P. putida macrorestriction fragments used as DNA probes were obtained from a 1.2% (wt/vol)
low-melting-point agarose PFGE gel by diluting the agarose with TE
buffer to a final concentration of 0.3% (wt/vol) agarose, melting it
at 68°C, and performing phenol, phenol-chloroform, and chloroform
extractions. Finally, DNA was ethanol precipitated according to
standard procedures (66). All probes were digoxigenin
labeled by Klenow random primer extension according to the recommended
procedure (DIG-DNA labeling and detection kit; Boehringer Mannheim).
Blotted filters were prehybridized, hybridized, washed, and
immunologically developed according to the supplier's instructions.
High-stringency conditions (50% [vol/vol] formamide and 42°C) were
used for P. putida gene probes. For heterologous gene
probes, the conditions were less stringent: the concentration of
formamide in the hybridization solution was decreased to 30%
(vol/vol), or the hybridization temperature was reduced to 28°C.
Blots were stored at 
20°C and reused several times.
Amplification of DNA by PCR.
Several probe templates were
prepared by PCR amplification of genomic DNA. Genomic DNA from P. putida was prepared as described before (63). P. putida sequences were obtained from the EMBL database and used to
design the following primers. For the cell division ftsZ
gene, the primers 5'-GGCCCCAGTGCTTGAACGCT-3' and 5'-TTAATCAGCCTGACGACGCA-3' were used; amplification yielded
a 1.3-kb fragment. The gene pyrB, encoding aspartate
carbamoyltransferase, was obtained after PCR amplification with the
primers 5'-TACTGATGGGCGGTCGCACC-3' and
5'-CCCGCTCATGGCCATGGACA-3'; a 1.2-kb fragment was obtained. The lipoamide dehydrogenase (lpdG) gene was obtained after
PCR amplification with primers 5'-GCCAGGTCGTGATTCGCCCG-3'
and 5'-CCCGCCGTGGTTTCTTATAA-3'; it yielded a 1.7-kb
fragment. To obtain the gene encoding muconate-lactonizing enzyme
(catB), primers 5'-GACAAGCGCGCTGATTGAAC-3' and
5'-ACAGCGACGGGCGAAGCGCG-3' were chosen. All PCR
amplifications were performed as recommended by the manufacturer in a
Perkin-Elmer DNA thermal cycler under the following conditions: 1 min
at 92°C, 1 min at 60°C, and 1 min at 72°C, except for
lpdG, for which the annealing temperature was 45°C.
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RESULTS |
Choice of restriction enzymes, separation of fragments, and
estimation of P. putida KT2440 genome size.
To
construct the physical map, the restriction enzymes chosen should be
able to generate a manageable number of fragments. Our selection of
appropriate rare-cutting endonucleases was based on the high G+C
content (62.5 mol%) of P. putida (42). Three enzymes with rich A+T recognition sequences, DraI,
SspI, and XbaI, were assayed. These 6-bp
recognition sequence enzymes generated more than 50 fragments each
(data not shown) and were considered inappropriate. SpeI,
which recognizes the rare tetranucleotide CTAG (35) within
its 6-bp recognition sequence, was also tested. It produced more than
40 fragments, many less than 50 kb in size, and thus was not suitable
for this study. PacI, SwaI, and PmeI, which recognize an A+T-rich 8-bp target sequence, generated 6, 19, and
9 fragments respectively (Fig. 1; Table
1). These enzymes were considered
suitable, as was I-CeuI, which recognizes a specific 26-bp
sequence within the gene encoding the 23S rRNA (30) and which cut the P. putida KT2440 genome in six fragments (Fig.
1; Table 1).
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FIG. 1.
CHEF electrophoresis of fragments of P. putida KT2440 genomic DNA predigested with different restriction
enzymes. All gels were 1.2% (wt/vol) agarose and were run in 0.5× TBE
buffer with the exception of gel A, which was a 0.8% (wt/vol) agarose
gel run in 1× TBE buffer. (A) Running conditions: 50 V for 216 h;
pulse times, 1,000 s for 24 h, followed by 1,000- to -4,000-s
linearly ramped pulse for 192 h. Running conditions: (B and C) 150 V for 70 h; pulse times, 200 s for 24 h, 120 s for
24 h, and 80 s for 22 h. In gel C, partial
I-CeuI fragments are indicated by arrows. (D and E) Running
conditions: 145 V for 66 h; pulse times, 120 s for 24 h,
70 s for 22 h, and 60 s for 20 h. (F) Running
conditions: 100 V for 72 h; 5- to 100-s linearly ramped pulse. (G)
Running conditions: 450 V for 4 h, pulse times, 0.5 s. Lanes:
S, SwaI; P, PmeI; Pa, PacI; C,
I-CeuI; Sp, SpeI; 6, SwaI plus PmeI; 7, PacI plus PmeI; 8, SwaI plus
PacI; 9, I-CeuI plus PmeI. DNA size
markers were phage  DNA concatemers (lane 1), intact phage DNA
plus phage DNA digested with HindIII (lanes 2), and
chromosomes of S. cerevisiae (lanes 3) and H. wingei (lanes 4). Lane 5 contains total P. putida
DOT-OX3 DNA digested with SwaI. Sizes are indicated in
kilobases.
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TABLE 1.
Fragment sizes of all single restriction enzyme digests
of SwaI, PmeI, I-CeuI, and
PacI of the P. putida KT2440 chromosome
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All DNA fragments generated by restriction were separated by CHEF
electrophoresis. Different running conditions were required for optimal
resolution in each size range (Fig. 1A through E). For example, a
period of 9 days was needed to resolve the largest fragment detected in
these analyses, i.e., the 2,850-kb I-CeuI fragment A (Fig.
1A). Fragments in the size range between 1,000 and 65 kb were resolved
under the running conditions specified in the legend to Fig. 1, so that
the size of each fragment could be accurately determined (Fig. 1B
through F). The smallest SwaI and PmeI
single-digest fragments, which were smaller than 50 kb, could be
detected by ethidium bromide staining (Fig. 1G). 32P-end
labeling of P. putida DNA digested previously with
SwaI or PmeI did not resolve any additional
fragments upon autoradiographic development (Fig.
2). Table 1 summarizes the sizes of the
restriction fragments obtained with the endonucleases used in this
work, averaged from more than 10 separate gels. The genome size of
P. putida KT2440 was estimated by adding the sizes of the
fragments generated by each of the endonucleases used; this yielded an
average genome size of 6.0 Mb (Table 1) with an error of less than 2%.
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FIG. 2.
Identification of genomic fragments of P. putida KT2440 smaller than 15 kb after digestion with
SwaI, PmeI, and SwaI plus
PmeI and 32P end labeling. Total DNA of P. putida KT2440 genomic DNA was digested with SwaI (lane
S), PmeI (lane P), and SwaI plus PmeI
(lane S/P) and then end labeled with 32P as described in
Materials and Methods. Fragments were separated in a conventional 0.8%
(wt/vol) agarose gel run in 1× TAE buffer for 3 h at 5 V
cm 1. The gel was exposed to Kodak photographic film and
developed. Size fragments are indicated in kilobases. Bands of interest
and their sizes are indicated by arrows.
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The size of the genome was further confirmed when the sizes of the
fragments resulting from double digestion with
SwaI-PmeI and PmeI-PacI
(Table 2) were added. 32P-end
labeling of P. putida DNA digested with SwaI and
PmeI revealed two additional fragments of 8 and 1 kb which
were not detectable by ethidium bromide staining (Fig. 2).
In the course of mapping, the identity of the fragment patterns between
successive plug preparations was checked. No change in fragment pattern
was observed during the period of study.
Construction of the physical map.
Two approaches were combined
to organize the restriction fragments into a map: (i) physical methods
and (ii) hybridization analysis.
(i) Physical methods.
In addition to total digestion of the
chromosomal DNA of P. putida with SwaI,
PacI, PmeI, and I-CeuI, we tried to
obtain partial digestions with these enzymes by reducing either the
amount of enzyme or the incubation time. For I-CeuI, two
partial fragments (Fig. 1C) of 750 and 470 kb were observed. These
fragments can correspond only to the combinations I-CeuI-D
(610 kb)-I-CeuI-F (126 kb) and I-CeuI-E (330 kb)-I-CeuI-F (126 kb), respectively. Therefore, these
results unequivocally link the fragments in the order
I-CeuI-D-I-CeuI-F-I-CeuI-E).
Partial digestion with SwaI generated a wide range of
fragments, but only two of them (420 and 220 kb) could be unequivocally assigned to the combinations SwaI-H (352 kb)-SwaI-L (65 kb) and SwaI-J (180 kb)-SwaI-N (30 kb) (not shown).
To extend the limited information derived from partial digestion of
chromosomal DNA, we analyzed the SwaI-PmeI double
digests of the P. putida KT2440 chromosome. This was
expected to provide information about linkage of the fragments
generated by SwaI and PmeI (Fig. 1B, D, and F).
Fragment SwaI-A remained uncut by PmeI and was
assumed to be contained within PmeI-A or PmeI-B.
Fragment SwaI-B included a PmeI restriction site
yielding a smaller fragment of 680 kb (Table 1). Fragments
SwaI-C and SwaI-D were both cut by
PmeI; one of them generated a 560-kb fragment (Fig. 1B), but the other fragment generated could not be identified unequivocally. SwaI-E and the double SwaI-G fragments,
SwaI-H, SwaI-I, SwaI-K', SwaI-L, and SwaI-M remained uncut by
PmeI (Table 2; Fig. 1D and F).
To further exploit this information, we used a modified version of
two-dimensional restriction fragment analysis as described by Bautsch
et al. (2) and Römling et al. (65). Total
chromosomal DNA was digested with PmeI, and the resulting
fragments (PmeI-A through PmeI-G) were initially
separated on a CHEF electrophoresis gel and identified (Fig.
3A). An agarose plug with each of the DNA
bands was then removed from the gel and further digested with SwaI, and the new fragments were separated in a second
electrophoresis (Fig. 3B). As a control, total chromosomal DNA of
P. putida KT2440 was digested with SwaI. These
analyses revealed that fragments SwaI-A, SwaI-H,
SwaI-I, SwaI-L, SwaI-M, and
SwaI-N were internal fragments of PmeI-A, and as
expected, two new fragments were generated by SwaI within
PmeI-A. These were called SP3 (450 kb) and SP12 (21 kb)
(Table 2). PmeI-B contained SwaI-E,
SwaI-K', and SwaI-G fragments and two new
fragments, designated SP9 (60 kb) and SP13 (8 kb) (Table 2).
PmeI-C generated fragments SP1 (680 kb) and SP8 (130 kb).
PmeI-D contained the SwaI-N fragment and yielded two new fragments called SP2 (560 kb) and SP6 (172 kb).
PmeI-E yielded SP4 (390 kb) and SP5 (330 kb) upon digestion
with SwaI, whereas PmeI-F contained
SwaI-O and yielded fragments SP7 (165 kb) and SP14 (1 kb).
Digestion of PmeI-G with SwaI resulted in fragments SP10 (45 kb) and SP11 (30 kb). This approach allowed us to
locate all of the SwaI PmeI-site-free fragments
within a PmeI fragment. Based on these data, we linked some
of the PmeI fragments to each other, on the assumption that
the SP fragments generated in the SwaI/PmeI
double digestion would equal the size of a previously determined
SwaI fragment. For example, fragments SP3 (450 kb) and SP8
(130 kb) were assumed to come from SwaI-D (584 kb) and
therefore to establish the linkage between PmeI-A and
PmeI-C. Similar examples linked fragments PmeI-F
with PmeI-E and PmeI-E with PmeI-B.
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FIG. 3.
Locations of SwaI restriction sites within
PmeI fragments. (A) Separation of P. putida
KT2440 PmeI fragments in the first electrophoresis; (B)
separation of SwaI fragments generated upon digestion of
different PmeI fragments. In gel A, two different lanes of
total DNA digested with PmeI were run side by side. After
ethidium bromide staining of one of them (A), the agarose plugs
containing the PmeI fragment of interest were excised and
digested with SwaI. Finally DNA fragments were separated in
the second electrophoresis (B). Lanes: 1, phage lambda DNA undigested
and digested with HindIII; 9, phage lambda DNA
concatemers: S, P. putida KT2440 genomic DNA digested with
SwaI. Lanes 2, 3, 4, 5, 6, 7, and 8 correspond to fragments
PmeI-A, PmeI-B, PmeI-C,
PmeI-D, PmeI-E, PmeI-F, and
PmeI-G, respectively, digested with SwaI. Sizes
are indicated in kilobases.
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The order of the SwaI fragments included in
PmeI-A could not be unequivocally assigned on the basis of
the available information. Useful information was derived from strain
DOT-OX3, a P. putida KT2440 mutant unable to synthesize the
O antigen of the lipopolysaccharide that had been generated after
mutagenesis with mini-Tn5' luxAB (Fig. 1D, lane 4). When we
tried to locate the position of the mini-Tn5 on the
chromosome of this strain, we found that fragments SwaI-A
and SwaI-H had disappeared and two new fragments, of 750 and
480 kb, had appeared. The sum of the sizes of these two new fragments
is similar to the sum of the sizes of SwaI-A and
SwaI-H (Fig. 1D). This finding unequivocally linked these
two fragments and suggested that this mutant must contain an inversion
of at least 150 kb.
In summary, the above series of analyses allowed us to establish that
SwaI-A, SwaI-H, and SwaI-L were
linked, as were SwaI-J and SwaI-N. We also found
that fragments E, F, and D resulting from I-CeuI digestion
were also linked. PmeI-A was linked to PmeI-C, whereas PmeI-E was linked to PmeI-F and
PmeI-B. The order of fragments in the intact chromosome was
PmeI-F,E,B. We identified a number of SwaI
fragments within each of the PmeI fragments. In all, the above pattern of fragment linkage provided a low-resolution map in
which approximately two-thirds of the chromosome backbones have been established.
(ii) Hybridization analysis.
To enhance the physical map, we
used Southern blot analysis of a series of P. putida KT2440
derivatives labeled with mini-Tn5-Km. This series of mutants
is called P. putida EEZ15K-x where x
is 3, 8, 12, 14, 16, 17, 22, 23, 24, or 30 (56). As a probe,
the Kmr determinant gene was used and the position of the
Kmr gene in P. putida EEZ15K-x was
established. Table 3 summarizes the
hybridization data obtained for the DNA of each mutant cut with
SwaI, PmeI, and I-CeuI. This analysis
confirmed the location of the different SwaI fragments
within the PmeI fragments. In addition, it revealed that
fragments I-CeuI-E, I-CeuI-F, and
I-CeuI-D were located within PmeI-A. We also
found that I-CeuI-C was contained within PmeI-A,
as surmised from evidence that the Kmr cassette in P. putida EEZ15K-30 was located in SwaI-A,
PmeI-A, and I-CeuI-C. The same was true for
EEZ15K-16 except that in this mutant the mini-Tn5 lies
within the SwaI-H fragment. The finding that the
Kmr cassette in mutant EEZ15K-23 was located in
SwaI-L and I-CeuI-E and that the
mini-Tn5 in mutant EEZ15K-14 was in SwaI-I and in I-CeuI-E linked SwaI-I with the set of fragments
SwaI-A-SwaI-H-SwaI-L.
Given that SwaI-D established the linkage between
PmeI-A and PmeI-C, the position of the
Kmr cassette in mutant EEZ15K-22 within fragments
SwaI-B, PmeI-C, and I-CeuI-B confirmed
the connection between fragments SwaI-D and
SwaI-B as well as that between fragments I-CeuI-D
and I-CeuI-B.
We deduced that the largest I-CeuI-A fragment included
PmeI-B, PmeI-D, and PmeI-E, because
mutants EEZ15K-8, EEZ15K-17, and EEZ15K-24 were located within these
fragments (Table 3). From the information thus obtained, the linkage of
all I-CeuI fragments was established as
I-CeuI-A,C,E,F,D,B.
In addition, the 51 gene probes listed in Table
4 were used against P. putida
KT2440 chromosomal DNA digested with SwaI, PmeI,
I-CeuI, PacI, SwaI-PmeI,
etc. (Tables 5 and
6). This allowed us to further define the
map. As an example, the hybridization gel with the npt gene
probe and the dnaJ-dnaB-carAB set of genes is shown in Fig.
4.
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TABLE 5.
Fragments restricted with SwaI,
PmeI I-CeuI, and PacI which gave a
hybridization signal with several gene probes
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TABLE 6.
Fragments restricted with
SwaI/I-CeuI, SwaI/PmeI,
I-CeuI/PmeI, PacI/PmeI, and
PacI/SwaI which gave a hybridization signal with
several gene probes
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FIG. 4.
Hybridization analysis with several gene probes.
P. putida KT2440 DNA was digested with SwaI
(lanes S), PmeI (lanes P), and SwaI plus
PmeI (lanes 1), and DNA fragments were separated by CHEF
electrophoresis. The three lanes on the left were separated from the
two other lanes, and each set of DNA was transferred to a nylon
membrane. In panel B, the three lanes on the left correspond to
hybridization with the npt gene probe, while the other two
lanes correspond to hybridization with the dnaJ-dapB-carAB
set of genes. Sizes are indicated in kilobases.
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Analysis of all hybridization assays unequivocally located all
I-CeuI and PacI fragments except fragments
PacI-E and PacI-F, which, because they showed the
same location within SwaI-C, PmeI-D, and
I-CeuI-A, could be exchanged with each other. We were able to locate most but not all SwaI and PmeI
fragments. The positions of the smaller SwaI
(SwaI-K through -O) and PmeI (PmeI-G through -I)
fragments remained undetermined. These fragments were separated with
PFGE, and fragments smaller than 130 kb were extracted and purified
from agarose gels (Table 6). These fragments were used directly as
probes against blots of chromosomal DNA digested with SwaI,
PmeI, and I-CeuI. A single SwaI or
PmeI fragment used as a probe should produce a single band
when hybridized against a filter carrying fragments of a digest
produced by the same enzyme and should produce one or more bands when
hybridized to a digest produced by one of the other enzymes. The data
from these experiments are summarized in Table
7. Figure 5 shows an example in which the
SwaI-J fragment was used as a probe. It hybridized with
itself (180 kb) and with the PmeI-D fragment (729 kb) in
single-digest assays and with the SP6 (172 kb) fragment in the
PmeI-SwaI double digest (Fig.
5). It also hybridized with the
I-CeuI fragment A (not shown). Similar assays allowed us to
unequivocally locate the SwaI-G, SwaI-J,
SwaI-K', and SwaI-O fragments, as well as the
PmeI-F, PmeI-G, PmeI-H, and
PmeI-I fragments (Table 7).
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FIG. 5.
Hybridization analysis with small chromosomal fragments.
Fragment SwaI-J was extracted from a gel and used as a probe
against P. putida KT2440 DNA digested with SwaI
(lane S), PmeI (lane P), or SwaI plus
PmeI (lane 1).
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Hybridization with fragments SwaI-L and SwaI-M
produced multiple bands with all of the enzymes tested; these bands
represent rrn operons within these fragments, as shown by
hybridization with the rrn gene probe (Fig.
6). Fragments SwaI-L and
SwaI-M were located on the basis of the positions of the
I-CeuI restriction sites and the hybridization data.
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FIG. 6.
Hybridization analysis with the rrs gene as a
probe. P. putida KT2440 genomic DNA was digested with
SwaI (lanes S), PmeI (lanes P), I-CeuI
(lanes C), SwaI plus PmeI (lanes 1), or
I-CeuI plus PmeI (lanes 2). The sizes (in
kilobases) indicated on the right correspond to S. cerevisiae chromosomes used as a size marker.
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Construction of the gene map.
Forty-five previously identified
genes or gene clusters were located on the P. putida KT2440
physical map (Tables 5 to 7; Fig. 7) by
probing chromosomal digests with available cloned genes and PCR
fragments. The gene probes used for this purpose are listed in Table 4.
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FIG. 7.
Physical and genetic map of P. putida KT2440.
The circular chromosome is represented as a series of overlapping
fragments for the enzymes SwaI (Sw), PmeI (Pm),
Pac (Pa), and I-CeuI (Ce). Genetic loci were
assigned to restriction fragments by Southern hybridization analysis;
groups of markers that hybridize at the same restriction fragments are
underlined. The exact positions of the six rDNA operons are
indicated.
|
|
The backbone of the chromosome is defined by the position of the
rrn loci rrs (16S), rrl (23S), and
rrf (5S) and their distribution with respect to the origin
of replication (5). I-CeuI recognizes a specific
26-bp sequence within the rrl gene. The mapped
I-CeuI restriction sites localized six rrn
operons, which we have designated rrnA through
-F, in the physical map of P. putida KT2440 (Fig. 7). A specific rrs probe was used to validate the existence
of an rrn operon within the SwaI and
PmeI fragments that contain the I-CeuI
restriction sites (Fig. 6). Hybridization of the rrs probe
to I-CeuI chromosomal digests gave no signal with fragment I-CeuI-B but gave a signal with the rest of the
I-CeuI fragments (Fig. 6).
Probes consisting of more than a single gene, such as
hemA-hemM-prf1, dnaJ-dapB-carAB, and
pcaG-pcaH, hybridized to a single band only, suggesting that
these genes are probably contiguous in P. putida KT2440. The
positions of the genes necessary for leucine, threonine, and arginine
biosynthesis were mapped by Southern hybridization with cosmid clones
from a P. fluorescens SBW25 genomic library (46).
Each clone hybridized to a single band under high-stringency conditions, showing that these species are similar in gene organization within these cosmid areas.
No homology was detected to several E. coli probes
(fabF, asmE, mobABCD, msrA,
and rpoE) or to pca-qui-pob from
Acinetobacter calcoaceticus, to bkdR from
P. putida PpG2, or to hemB from P. aeruginosa, even under low-stringency conditions.
| |
DISCUSSION |
We have generated the first complete physical map for the enzymes
I-CeuI, PacI, PmeI, and
SwaI in P. putida KT2440. A single chromosome was
shown to be circular and to have an estimated size of 6.0 Mb. Two
independent mapping approaches, (i) analysis of fragments resulting
from digestion of the whole genome with rare-cutting restriction
enzymes and (ii) Southern hybridization, were used to minimize possible
errors and to validate the data obtained separately with each approach.
A similar method was used to construct the maps of Haemophilus
influenzae (29) and Mycoplasma mycoides (44). In the P. putida KT2440 map, a total of 40 restriction sites (6 I-CeuI, 6 PacI, 9 PmeI, and 19 SwaI) were positioned on the map,
achieving an average resolution of approximately 160 kb. Our procedure
ensures that DNA fragments larger than 18 kb in size were detected by
ethidium bromide staining of whole genome DNA and that the use of
32P-labeled fragments detected fragments of up to 1 kb. It
is unlikely that we overlooked fragments smaller than 1 kb because we
avoided overrunning the gels when fragments were labeled with
32P. The genome of this P. putida strain is only
100 kb larger than the 5.9-Mb P. aeruginosa PAO1 genome
(65), 1.78 Mb larger than the average Pseudomonas
stutzeri genome (14), about 400 kb larger than the
P. syringae pv. phaseolica genome (7), and about
600 kb smaller than the 6.63-Mb P. fluorescens SWB25 genome
(46).
A partial genetic map of the P. putida KT2440 genome was
obtained by Southern hybridization with 51 probes (Table 4), of which
38 gave positive signals locating a total of 63 genes (Fig. 7),
including key markers such as oriC, recA,
gyrB, rpoS, rpoN, rpoD, and
the rDNA operons. Figure 7 summarizes the results obtained for more
than 100 gels resulting from single or double digestion with
rare-cutting enzymes in a large series of hybridization experiments. Several auxotrophic markers (leu, thr, and
arg) were positioned, as were genes involved in pilus
biosynthesis and motility, lipopolysaccharide production, and inorganic
nitrogen assimilation. In addition, we located several catabolic
operons (genes) for the metabolism of aromatic compounds. P. putida KT2440 is a nonaggressive root colonizer (37).
The genes involved in C4-dicarboxylate transport (dctA, -B, and -D) are important for
rhizosphere colonization (37). In this study, we located the
dctA gene.
The distribution of signals allowed us to identify a genetically dense
region around oriC; the rrn operons seem to be
grouped in this region with five of the six operons occupying one-third of the genetic map (Fig. 7). P. putida contains six rRNA
loci, designated rrnA through -F. Localization of
the I-CeuI sites precisely positioned the rrn
operons (30). Assuming the 5'-16S-23S-3' orientation, the
hybridization data with a 16S rRNA probe suggested that the rDNA genes
are organized in a typical eubacterial manner, with the rrn
operons transcribed divergently. The six copies of rrn
operons in P. putida KT2440 contrast with the five
rrn operons in P. fluorescens SBW25
(46) and P. syringae pv. phaseolica (7) and the four described for P. aeruginosa PAO1
(65). Among eubacteria, the number of rRNA operons varies
between 14 in Clostridium beijerinkii (75) and 1 in some species of the genus Mycobacterium (28).
This finding suggests different patterns of gene rearrangements not
only among different genera but also within the same genus.
This information provides a basis for comparing different members of
the genus Pseudomonas. Figure
8 shows the locations of the common
genetic markers in P. putida KT2440, P. fluorescens SBW25, and P. aeruginosa PAO1. It is
accepted that translocatable elements allow bacterial chromosomes to
acquire new genes by lateral transfer from other species, so that a
different map location for genes performing the same function in
species derived from a common ancestor does not necessarily reflect a
chromosomal rearrangement since the time of divergence, but may mean
that the two species have independently acquired that gene
(27). However, the presumed ancestor of P. aeruginosa, P. putida, and P. fluorescens
probably already possessed all or many of the genes required for DNA
recombination, metabolism of simple metabolites, and uptake of
compounds abundant in the environment (such as dicarboxylic acids), and
so these markers should be reliable indicators of chromosomal
rearrangements. The limited number of hybridization experiments with
cosmids derived from P. fluorescens suggests that at least
in the cases we studied, clusters of genes were conserved, although our
limited information does not allow us to draw definitive conclusions. A
detailed comparison of the genomic organization of different
pseudomonads thus awaits further experimental analysis.
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|
FIG. 8.
Comparison of the maps of P. fluorescens
SBW25, P. putida KT2440, and P. aeruginosa PAO1.
To facilitate comparisons, the circular maps were opened at an
arbitrarily chosen point and positioned with respect to the origin of
replication. Only the genes found in both P. putida and one
of the other bacteria are shown, connected by a dashed line. The
position of each gene is expressed as a ratio of the position of that
gene relative to oriC.
|
|
Determination of the physical and partial genetic map of the P. putida genome constitutes a significant step forward in terms of
comparative genome analysis and will aid the genome sequencing project.
The map provides a sound framework for studies of the taxonomy of
Pseudomonas species as well as for studies of the colonization of different niches and the utilization of different nutrients by these bacteria.
This work was supported by grants from the European Commission
(BIO4-CT97-2183) and CICYT (BIO 97-0641).
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Abstract
Introduction
