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Journal of Bacteriology, January 2000, p. 91-99, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Proline Catabolism by Pseudomonas putida: Cloning,
Characterization, and Expression of the put Genes in the
Presence of Root Exudates
Susana
Vílchez,1
Lázaro
Molina,2
Cayo
Ramos,3 and
Juan L.
Ramos1,*
Department of Biochemistry and Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas,1 and GX-Biosystems
España, Pinos Genil,2 Granada, Spain,
and Department of Microbiology, Technical University of
Denmark, Lyngby, Denmark3
Received 15 June 1999/Accepted 11 October 1999
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ABSTRACT |
Pseudomonas putida KT2442 is a root-colonizing strain
which can use proline, one of the major components in root exudates, as
its sole carbon and nitrogen source. A P. putida mutant
unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The
mini-Tn5 insertion was located at the putA
gene, which is adjacent to and divergent from the putP
gene. The putA gene codes for a protein of 1,315 amino acid
residues which is homologous to the PutA protein of Escherichia
coli, Salmonella enterica serovar Typhimurium,
Rhodobacter capsulatus, and several Rhizobium
strains. The central part of P. putida PutA
showed homology to the proline dehydrogenase of Saccharomyces
cerevisiae and Drosophila melanogaster, whereas the
C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde
dehydrogenases. This suggests that in P. putida, both
enzymatic steps for proline conversion to glutamic acid are catalyzed
by a single polypeptide. The putP gene was homologous to
the putP genes of several prokaryotic microorganisms, and
its gene product is an integral inner-membrane protein involved in the
uptake of proline. The expression of both genes was induced by proline
added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both
putA and putP genes was maximal and proline
independent. Corn root exudates collected during 7 days also strongly
induced the P. putida put genes, as determined by using
fusions of the put promoters to 'lacZ. The
induction ratio for the putA promoter (about 20-fold) was
6-fold higher than the induction ratio for the putP promoter.
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INTRODUCTION |
Pseudomonas putida KT2442
is an efficient root colonizer in a number of agriculturally important
plants. In field assays, the root colonization of corn and broad bean
by this P. putida strain ranged from about 105
to 107 CFU per g of soil, depending on the year and the
season (38, 39). However, in soils without plants, the
number of viable cells never surpassed 103 CFU per g of
soil (39) and frequently remained at a level below 102 CFU per g of soil. Little is known about the nature of
the nutrient source available for this strain during root colonization.
Amino acids present in plant exudates may help satisfy the energy
demands of rhizobacteria (25). Our group and others have
identified the amino acids present in the root exudates of corn plants.
Almost all of the 20 amino acids most frequently present in the
proteins can be detected, with proline one of the most abundant
(4, 8, 29, 41, 56; C. Ramos and L. Molina,
unpublished results). These observations raise the possibility that, at
least in the corn root rhizosphere, proline catabolism may play a
relevant role in supporting root colonization. Nevertheless,
information regarding proline catabolism by Pseudomonas
strains is scarce (34, 35).
The first step for proline catabolism requires the entry of this amino
acid into the cells (60). In enteric bacteria, proline is
taken up by several transport systems that differ in their Vmax and affinity for proline. The PutP protein
represents the major proline uptake system in Escherichia
coli and Salmonella spp., with a
Km of about 2 µM (61). The uptake
of proline via PutP is coupled to the entry of sodium ions (7, 10,
26, 47, 60).
Proline is converted into glutamate in a two-step process carried
out by proline dehydrogenase (PDH) (EC 1.5.99.8) and
pyrroline-5-carboxylate dehydrogenase (P5CDH) (EC 1.5.1.12)
(21, 33, 59). In eukaryotes, PDH and P5CDH are encoded by
two different genes (30, 58), while in enteric bacteria
(2, 31, 63), Rhodobacter capsulatus (27), Rhizobium meliloti (25), and
Bradyrhizobium japonicum (53), both steps for
proline utilization are catalyzed by a single polypeptide encoded by
the putA gene. In addition to these enzymatic activities,
the PutA protein, at least in enterobacteria, is involved in the
transcriptional control of the put genes. It seems that PutA
functions as a repressor, inhibiting expression from the divergent
put genes (33, 44).
In the present study, we isolated a P. putida KT2442 mutant
unable to use proline as its sole C and N source. The mutation was
complemented by using a P. putida cosmid library, and we
rescued and analyzed the complete nucleotide sequence of the P. putida put genes. We also show that put gene expression
in this strain is inducible by proline present in root exudates.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and culture conditions.
P.
putida KT2442 was described in an earlier publication
(18). It can use benzoate as its sole C source and exhibits
resistance to rifampin, chloramphenicol, and ampicillin. Strain S14D2
is a KT2242 mutant unable to use proline as its sole C and N source (Table 1). The E. coli strains
used in this study are shown in Table 1.
Bacterial cells were grown in Luria-Bertani medium or minimal M9 medium
with succinate (20 mM) and/or proline (20 mM) as a
C source
(
1). When proline (20 mM) was used as the sole C and
N
source, M9 depleted of ammonium, called M8, was used. When necessary,
ampicillin, chloramphenicol, kanamycin, rifampin, and tetracycline
were
added to final concentrations of 100, 30, 25, 10, and 10
µg/ml,
respectively.
DNA techniques.
Plasmid DNA was isolated by the alkaline
lysis method with the QIAprep spin plasmid minipreps kit (Qiagen
catalog no. 27104). Total DNA was isolated by modifying the method of
Kado and Liu as described by Ramos-González et al.
(46), except that the 30-min incubation step at 55°C was
omitted. DNA digestions with restriction enzymes, ligations, and
transformations were performed by standard procedures (48).
DNA in both strands was sequenced with the dideoxy sequencing method,
using the ABI Prism dRhodamine terminator kit (reference
no. 403042;
Perkin-Elmer).
Southern hybridization and DNA labeling.
DNA fragments were
separated in agarose gels and transferred onto nylon membranes by
capillary blotting as previously described (48). Specific
probes for hybridization were recovered from agarose gels with an
agarose gel DNA extraction kit (reference no. 1696505; Boehringer
Mannheim). All probes were labeled with digoxigenin by Klenow random
primer extension according to the recommended procedure (3).
Blotted filters were prehybridized, hybridized, washed, and
immunologically developed according to the supplier's instructions.
High-stringency conditions (50% [vol/vol] formamide at 42°C) were used.
Mutagenesis of P. putida by the mini-Tn5
luxAB-Km transposon.
Triparental matings involving P. putida KT2442 as the recipient, E. coli
CC118
pir(pCK220) as the transposon donor strain (52), and E. coli HB101(pRK600) as the helper
strain were carried out as described by de Lorenzo and Timmis
(16). Transconjugants of P. putida were selected
on M9 minimal medium plates with 5 mM benzoic acid as the sole C source
and supplemented with kanamycin and rifampin. About 5,000 independent
clones were tested for their ability to grow on M8 minimal medium with
proline as the sole C and N source. Four mutants unable to produce
colonies on minimal medium with proline were kept for further studies.
Complementation assays.
The pCRR831 cosmid (Table 1) (C. Ramos and L. Molina, unpublished results) selected from a P. putida KT2442 gene bank (M. I. Ramos-González,
unpublished data) was used for complementation studies. pCRR831 was
transferred by conjugation by the filter-mating technique
(16) to the P. putida S14D2 mutant unable to grow with proline as the sole C and N source. Filters with a mixture of
donor [E. coli HB101(pCRR831)], recipient (P. putida S14D2), and helper [E. coli HB101(pRK600)]
strains at a ratio of 1:5:1 were incubated for 4 h at room
temperature on Luria-Bertani plates. The cells were suspended in 1 ml
of M9 minimal medium, and 100 µl was plated on selective minimal
medium (M9 minimal medium with 10 mM benzoic acid, 10 µg of rifampin
per ml, and 10 µg of tetracycline per ml). The transconjugants
obtained were tested for their ability to grow on proline as the sole C
and N source.
Enzyme assay.
P. putida cells were grown on succinate,
proline, or succinate plus proline as the sole C source. Cells were
harvested by centrifugation, resuspended in a Tris buffer (pH 7.0; 100 mM), and permeabilized with toluene by vortexing. PDH activity was measured at 30°C in a 7-ml reaction mixture that contained 100 µmol
of Tris buffer (pH 7.0), 45 µmol of proline, and 4.5 µmol of
o-aminobenzaldehyde. The
1-pyrroline-5'-carboxylic acid (P5C) that formed reacted
with o-aminobenzaldehyde to produce a complex that exhibited
maximal absorbance at 443 nm (17). The absorbance was
corrected with a blank consisting of the same reaction mixture with
water instead of proline. PDH activity was expressed as the number of
nanomoles of P5C formed per milligram of protein.
Protein concentration in the cell extracts was determined with the
Bradford reagent (Bio-Rad reference no. 500.0006; Bio-Rad,
Madrid,
Spain) with bovine serum albumin as the
standard.
Collection of corn root exudates.
Seeds were germinated on a
sterile petri dish with water-agar. Seedlings were transferred to a
grid, and the hair root was submerged into a sterile solution of M9
medium without ammonium. After 7 days, the seeds were removed, and the
solution was filtered through a 0.2-µm sterile nitrocellulose filter
and stored at 
20°C until use. Proline concentrations in these
exudates ranged between 50 and 100 µM.
Construction of
PputA::lacZ and
PputP::lacZ fusions.
The divergent putA and putP promoter region was
amplified by PCR from total chromosomal DNA of P. putida KT2442 with primers 5'-TTACGAATTCCGATGTAGATCACGAAGG-3'
and 5'-TTACGGAATTCTGCTTTGAGTCGCTCACGG-3', which are
provided with a restriction site for EcoRI. Upon
amplification, as recommended by Ausubel et al. (3), DNA was
restricted with EcoRI and ligated to plasmid pMP220 digested
with EcoRI, so that transcriptional fusions of the
putA or putP promoters to a promoterless 'lacZ gene were generated. The nature of the fusion can be
distinguished by PCR amplification with an oligonucleotide primer based
on the lacZ sequence and on putA- or
putP-based primers, which result in a 0.8-kb fragment. The
plasmid bearing the
PputA::'lacZ fusion was
named pMIS5, and the one bearing the
PputP::'lacZ fusion was called pMIS12. The fusions were further confirmed by sequencing the
whole promoter region and the first 20 codons of the 'lacZ gene.
-Galactosidase activity was measured in
P. putida KT2440
and in
P. putida S14D2 bearing pMIS5 or pMIS12 and grown on
M9 minimal
medium with 20 mM succinic acid in the absence or the
presence
of 20 mM proline. Activity was determined according to
Gallegos
et al. (
19), and activity was given in Miller units
(
36).
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RESULTS |
Growth of P. putida KT2442 on proline as the sole C and
N source and isolation of mutants unable to metabolize proline.
We
first tested whether P. putida KT2442 was able to use
proline as the sole source of C, N, or both nutrients. This strain was
grown on M9 minimal medium with succinate as the sole C source. The
culture was diluted 100-fold into M8 minimal medium with 20 mM proline
and 10 mM NH4Cl (proline as the sole C source), 20 mM
succinate and 20 mM proline (proline as the sole N source), and 20 mM
proline (proline as the C and N source). The strain grew exponentially
with generation times of 1.70, 1.44, and 2.27 h when proline was
used as the sole C, N, and C plus N source, respectively.
We then mated
P. putida KT2442 with
E. coli
CC118
pir(pCK220) as described in Materials and Methods, and four
mutants defective
in proline utilization, called S14D2, S14D11, S15D3,
and S16D2,
were
found.
To further confirm this initial selection, growth of the strains was
tested in liquid M8 minimal medium with proline as the
sole C and N
source. Mutant S14D2 did not grow on minimal medium
after prolonged
incubation (Fig.
1), whereas the other
three mutants
did grow, although they had a very long lag period before
growth
started. See Fig.
1 for mutant S14D11. We measured the PDH
activity
of the wild-type and the mutant strains growing on M9 with
succinate
or succinate plus proline. The results obtained are shown in
Table
2. Neither the wild-type nor the
mutant strains exhibited any
statistically significant activity when
grown on succinate alone,
but the wild-type had high activity levels
when it grew in the
presence of proline. Mutants S14D11, S15D3, and
S16D2 also had
high levels of PDH activity when grown in the presence
of proline
(results not shown). In contrast, mutant S14D2 showed no
activity
when cells were grown on M9 with succinate and proline (Table
2). On the basis of these results, we considered S14D2 a true
proline
utilization-deficient strain, and it was retained for
further studies.
The other three mutants (S14D11, S15D3, and S16D2)
were discarded.
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FIG. 1.
Growth on minimal medium with proline as the sole C and
N source of the wild-type P. putida KT2442 and its mutant
strains deficient in proline utilization. Growth was monitored as an
increase in turbidity of the culture.  , P. putida
KT2442;  , P. putida S14D2;  , P. putida
S14D11;  , P. putida S14D2(pCRR831).
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Complementation of mutant S14D2 by pCRR831, cloning, and sequencing
of the put genes.
A P. putida KT2442 gene
bank constructed in the broad-host-range pLAFR3 cosmid (M. I. Ramos-González, unpublished data) was used to complement E. coli RM2 (Table 1), a mutant unable to grow on proline because of
a deletion of the putA and putP genes (20). A plasmid called pCRR831 was found to restore the
ability to use proline as the sole C and N source to the E. coli mutant strain (C. Ramos and L. Molina, unpublished results).
We transferred the Tcr pCRR831 plasmid to P. putida S14D2 and selected Kmr Tcr
transconjugants able to grow on M8 minimal medium with proline as the
sole C source. The frequency of appearance of transconjugants was
10
5 per recipient, and 100% of the transconjugants were
able to grow on M8 liquid medium with proline as the sole C and N
source. Figure 1 shows the growth of one random P. putida
S14D2(pCRR831) clone, compared with the growth of the wild type and the
mutant S14D2. This finding suggests that pCRR831 carries the proline
degradation genes. To corroborate this finding, we determined the PDH
activity of P. putida S14D2(pCRR831) growing on succinate or
succinate plus proline. As expected, pCRR831 restored this activity in
the mutant strain to levels similar to those found in the wild-type strain, when cells grew in the presence of proline (Table 2).
To locate the
put genes in pCRR831, cosmid DNA was digested
with
PstI and hybridized against the 4.2-kb
MluI
fragment of plasmid
pPC6 (
20), which carries the
putA
putP genes of
Salmonella enterica serovar Typhimurium.
The
P. putida put genes were located within
two
PstI fragments of 4.3 and 2.0 kb, which were subcloned in
pUC19 to yield plasmids pLCR12 and pLCR4, respectively (Fig.
2).
The DNA in both
PstI
fragments was sequenced on both strands.
The DNA sequences were
compared with those deposited in the GenBank
database, and the analysis
revealed that the 4.3-kb DNA fragment
bore the whole
putP
gene (1,479 bp), part of the '
putA' gene (450
bp), and the
intergenic region between
putP and
putA (355 bp).
These genes were transcribed divergently. Plasmid pLCR4, bearing
a 2-kb
insert of the
P. putida genome, also contained part of
the
putA gene; however, the translated DNA sequence did not
exhibit
a stop codon, nor did it account for the expected size of the
PutA protein when compared with the PutA sequences deposited in
GenBank. To complete the
putA gene, a 12-kb
HindIII fragment of
pCRR831 was subcloned in pUC19 to
yield pSLH4 (Fig.
2). DNA was
sequenced with specific 20-mer primers,
based on available
P. putida putA sequences, until the
complete
putA gene sequence was
obtained (3,948 bp). In all,
the
putA and
putP genes and the intergenic
region
covered 5,757 bp. The DNA sequence is available from GenBank
under
accession no.
AF153207. Downstream of both coding sequences,
stem-loop transcription terminator sequences were found, which
suggests that each gene makes a monocistronic mRNA.
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FIG. 2.
Localization of put genes of P. putida KT2442 in vectors pLCR4, pLCR12, and pSLH4. The pLCR4
plasmid contains 2 kb of the putA gene, and pLCR12 contains
450 bp of the putA gene, all the putP genes, and
the intervening regulatory region between the two genes that are
transcribed divergently. A 12-kb insert in plasmid pSLH4 bears the
complete proline utilization operon.
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The insertion of the mini-Tn
5 'luxAB-Km transposon in the
genome of
P. putida S14D2 was first located within the
putA gene,
based on hybridization assays. The region
surrounding the mini-Tn
5 was PCR amplified and the insertion
was specifically identified
at nucleotide 1635 of the
putA
gene
sequence.
Analysis of putA and putP gene
products.
The putA gene yielded the predicted PutA
protein, which is 1,315 amino acids long and shows homology to PutA
from different organisms such as Klebsiella aerogenes (71%
identity) (54), Salmonella serovar Typhimurium
(69% identity) (2), E. coli (69% identity)
(31), R. meliloti (54% identity)
(25), and B. japonicum (42% identity)
(53). The highest homology was the domain involved in PDH
activity (amino acids 337 to 588 in the P. putida PutA
protein) (Fig. 3). Within this domain, a flavin adenine
dinucleotide-binding pocket (residues 312 to 354) was identified. This
domain exhibited homology with PDHs from Saccharomyces cerevisiae and Drosophila melanogaster and therefore
seems to be involved in the conversion of proline to P5C, which
equilibrates in solution with glutamic acid semialdehyde.
According to Ling et al. (
31), amino acids 641 to 1074 are
required for P5CDH activity. An NADPH pocket (residues 850 to
857) with
the sequence FTGSTEVG was found within this region (
31),
which is highly similar to the corresponding PutA region in
E. coli and
Salmonella serovar Typhimurium (Fig.
3). This domain
exhibited
homology with aldehyde dehydrogenases, i.e., methylmalonate
dehydrogenase, betaine dehydrogenase, and 2-hydroxymuconic acid
semialdehyde dehydrogenase (
9,
11,
13,
42,
45,
51).
This
finding suggests that the real substrate of this activity
of PutA is
glutamic acid semialdehyde.
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FIG. 3.
Sequence alignment of PutA proteins of
prokaryotic origin. The strains and sources of the protein sequences
were as follows: P. putida (this study); E. coli
(31); Salmonella serovar Typhimurium
(2); K. aerogenses (54);
Agrobacterium tumefaciens (14); R. capsulatus (27); S. meliloti
(25); B. japonicum (53). The ALIGN
program was used. If the residue is identical in all the aligned
proteins, it appears printed on a black background. If the residue is
identical in 50% of the aligned proteins, it appears on a gray
background. The amino acid chosen for the consensus was present at the
given position in at least 50% of the aligned sequences. The PDH
domain, residues 337 to 588, is shown in a grey box, and the P5CDH
domain, residues 641 to 1074, is also boxed.
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A third region with high homology between PutA proteins but of unknown
function is located between amino acids 78 and 190.
In
E. coli, the PutA protein is able to associate with the cell
membranes. Three hydrophobic segments between residues 158 and
167, 767 and 817, and 1205 to 1220 may be important for such
interactions.
These segments are present in the
P. putida
PutA protein. In general,
the interdomains were less conserved (Fig.
3).
The
P. putida PutP protein is 493 amino acids long and
exhibits 85% similarity with PutP from
Pseudomonas
fluorescens, 76%
with
Salmonella serovar Typhimurium,
and 78% with
E. coli. The
Scamprosite program predicted 12 transmembrane segments for the
P. putida PutP protein, and
multiple alignments revealed extended
homology with PutP from other
sources that corresponded to transmembrane
segments (Fig.
4), whereas cytoplasmic and periplasmic
loops were
less well conserved. In addition, PutP presents homology to
transport
systems that are involved in the uptake of chemicals related
to
sodium entry, i.e.,
E. coli porter systems for inositol,
phenylacetic
acid, and pantothenate (
7,
15,
49,
55,
58).
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FIG. 4.
Sequence alignment of PutP proteins. Strains and sources
of the sequences were as follows: P. putida (this study);
Bacillus subtilis (64); P. fluorescens
(23); E. coli (40);
Salmonella serovar Typhimurium (37);
Rickettsia typhi (40); and Haemophilus
influenzae and Helicobacter pylori (55). The
ALIGN program was used. Other details are as in the legend for Fig.
3.
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Expression from the putA and putP gene
promoters.
To determine the expression of the put
genes, the divergent put promoter region was fused in a
broad-host-range vector to 'lacZ as described in Materials
and Methods to generate transcriptional fusions yielding pMIS5 and
pMIS12. These plasmids were transferred to the wild-type P. putida KT2442 and to its mutant P. putida S14D2.
-Galactosidase (LacZ) activity in P. putida KT2442 with one of these plasmids was measured in cells growing on minimal medium
with succinate and succinate plus proline under highly aerated
conditions. In wild-type cells growing on succinate, basal activity
from PputP (700 Miller units) was twofold higher
than for PputA (350 Miller units) (Table
3). In the presence of proline, the
increase in activity was 4- and 20-fold for the
PputP fusion and the
PputA fusion, respectively (Table 3). These
results suggest that the genes for proline catabolism are inducible.
Expression of the
putA and
putP genes was also
measured in the S14D2 mutant strain bearing pMIS5 or pMIS12 in cells
growing
on M9 minimal medium with succinate or with proline. In both
the
absence and the presence of proline, high levels of expression
were
found, about 2,700 Miller units for the
PputP::'
lacZ fusion
and
about 8,000 Miller units for the
PputA fusion.
These results
suggest that the PutA protein is involved in the
control of expression
from the
putA and
putP gene
promoters.
Induction of the Pput promoters by
corn root exudates.
P. putida KT2442 bearing plasmid
pMIS5 or pMIS12 was grown on minimal medium with succinate as the
sole C source until the mid-exponential growth phase was reached.
Cells were then either harvested and suspended in M8 minimal
medium without a C source or suspended in 7-day-old root exudates. The
suspensions were incubated at room temperature without agitation for 30 min to follow induction from the put promoters. The level of
-galactosidase activity from PputA and
PputP when cells were incubated in the presence
of corn root exudates was around 20- and 4-fold higher than the basal
level (Table 4). This suggests that
proline present in root exudates was able to promote expression of the
P. putida put catabolic genes.
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DISCUSSION |
Recent studies have focused their attention on the possible role
of amino acids as carbon substrates to support growth of microorganisms
in the rhizosphere of plants (24, 28, 63, 65). Proline has
been found to be a major compound in the corn root exudates; therefore,
this amino acid could be an important energy source for bacteria during
the first stages of colonization of the roots of plants. How deficiency
in the utilization of proline or other amino acids affects rhizosphere
colonization has not yet been studied in detail, although an R. meliloti mutant altered in proline catabolism exhibited reduced
ability to colonize the alfalfa root (25).
In this work we have approached the study of proline utilization in
P. putida, for which we isolated mutants unable to use proline as their C or N source. P. putida S14D2 was
considered a true proline utilization-deficient mutant because it did
not grow with proline, in contrast with other mutants isolated in this
study that showed retarded growth on proline. We found that in the
S14D2 mutant strain, the mini-Tn5 transposon was inserted in
the chromosome within a gene involved in proline catabolism (putA). Analysis of the P. putida putA gene
product revealed a domain structure similar to that of enteric bacteria
such as R. capsulatus and B. japonicum in which
the two steps for proline degradation to glutamate are catalyzed by a
single bifunctional dehydrogenase enzyme (2, 25, 27, 31, 51,
53). Analysis of the P. putida PutP protein suggests
that it is an integral inner-membrane protein that belongs to the
family of Na+ substrate symporters (15, 49, 58,
60). We showed that the putA gene is adjacent to the
putP gene and that these genes are transcribed divergently,
as is the case for enteric bacteria.
In P. putida, the putA and putP genes
seem to be regulated at the transcriptional level, with proline
either
supplied in culture medium or in root exudatesacting as an inducer,
as the expression from the putA and putP gene
promoters increased by about 20- and 4-fold, respectively, in the
presence of proline. In a putA mutant background, high
levels of expression from these genes occurred, suggesting that the
P. putida PutA protein acts as a repressor of
putA and putP gene expression, as also described
for enteric bacteria (11, 44). The fact that proline
metabolism in the soil bacterium P. putida is regulated by a
mechanism similar in principle to that of enteric bacteria is rather
surprising in the light of the differences in the ecological habitats
of these organisms. These similarities in the regulation of the
put genes in enteric bacteria and in Pseudomonas
prompted us to compare the intergenic regions between
putA and putP in these microorganisms. Figure
5 shows an alignment of the intergenic
region between putA and putP of
Salmonella serovar Typhimurium, E. coli, K. aerogenes and P. putida, from which it can
be seen that this region is 63 to 65 bp longer in enteric bacteria than
in P. putida, with a very large gap (28 nt) being observed
near the ATG start codon of the putP gene. In all four
microorganisms, putA and putP genes are
transcribed divergently, although differences in the location of
promoters are known, with overlapping promoters in
Salmonella serovar Typhimurium and well-separated
transcription starts in K. aerogenes and P. putida (12, 44; S. Vílchez and J. L. Ramos, unpublished results). In Salmonella serovar
Typhimurium, the intergenic putA-putP DNA is
intrinsically curved and it has been found that up to five segments
(marked in Fig. 5 by a line above the sequence) could be bound by
purified PutA protein. In enteric bacteria, it has been suggested that
the integration host factor plays a role in the expression from
putA and putP, and two sites (positions 1 to 13 and 330 to 344) (Fig. 5) in the Salmonella serovar
Typhimurium promoter region were found (6, 43, 44). Those
sites are not well conserved in the corresponding aligned sequence in
P. putida, and at present, we cannot predict whether or not
integration host factor plays a role in the transcription of the
put genes in the soil bacterium P. putida.
View larger version (48K):
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|
FIG. 5.
Alignment of the putA and putP
intergenic regions of enteric bacteria and P. putida. The
alignment includes the region between the start codons of
putA and putP. Gaps were introduced to allow
maximal scoring in the alignment with identical positions being shown
in boldface. The overlined bases indicate putative PutA binding
sites.
|
|
Therefore, we can conclude that although the pattern of gene control of
the putA and putP genes is similar in enteric
bacteria and in the soil-borne P. putida KT2440, the
molecular mechanisms of control may be very distinct.
| |
ACKNOWLEDGMENTS |
Susana Vílchez and Lázaro Molina contributed equally
to the experimental work.
Part of this study was supported by a grant from the European
Commission (BIO4-CT98-0283).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular and Cellular Biology of Plants,
Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científicas, Calle Profesor Albareda 1, E-18008
Granada, Spain. Phone: 34-958-121011. Fax: 34-958-129600. E-mail:
jlramos{at}eez.csic.es.
| |
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Abstract
Introduction
