Departamento de Biotecnología
Microbiana, Centro Nacional de Biotecnología, CSIC, Campus de
la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,
Spain,1 and Institute of
Biotechnology, ETH Hönggerberg, CH-8093 Zürich,
Switzerland2
In many microorganisms the first step for alkane degradation is the
terminal oxidation of the molecule by an alkane hydroxylase. We report
the characterization of a gene coding for an alkane hydroxylase in a
Burkholderia cepacia strain isolated from an oil-contaminated site. The protein encoded showed similarity to other
known or predicted bacterial alkane hydroxylases, although it clustered
on a separate branch together with the predicted alkane hydroxylase of
a Mycobacterium tuberculosis strain. Introduction of the
cloned B. cepacia gene into an alkane hydroxylase
knockout mutant of Pseudomonas fluorescens CHAO restored
its ability to grow on alkanes, which confirms that the gene analyzed
encodes a functional alkane hydroxylase. The gene, which was named
alkB, is not linked to other genes of the alkane
oxidation pathway. Its promoter was identified, and its expression was
analyzed under different growth conditions. Transcription was induced
by alkanes of chain lengths containing 12 to at least 30 carbon atoms
as well as by alkanols. Although the gene was efficiently expressed during exponential growth, transcription increased about fivefold when
cells approached stationary phase, a characteristic not shared by the
few alkane degraders whose regulation has been studied. Expression of
the alkB gene was under carbon catabolite repression when cells were cultured in the presence of several organic acids and
sugars or in a complex (rich) medium. The catabolic repression process
showed several characteristics that are clearly different from what has
been observed in other alkane degradation pathways.
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INTRODUCTION |
Alkanes are major components of
crude oil and are also present in many organisms, such as green plants
(43, 54). It has long been recognized that many
microorganisms can use medium- or long-chain n-alkanes as
sources of carbon and energy (27), which has stimulated
many studies on the usefulness of these organisms in the bioremediation
of oil spills and contaminated sites (reviewed in references
2 and 44). In addition, the enzymes involved in alkane degradation have proven to be useful and versatile
biocatalysts, opening the possibility of their use in the industrial
production of fine chemicals (16, 57, 59).
Alkane degradation by bacteria usually occurs through the sequential
oxidation of one or both terminal methyl groups of the molecule, first
to an alcohol, then to an aldehyde, and finally to a fatty acid, which
is assimilated through the 
-oxidation pathway (reviewed in
references 7 and 43). The best-characterized alkane degradation pathway is that encoded by the OCT plasmid of
Pseudomonas putida (Pseudomonas oleovorans) GPo1
(reviewed in references 56 and 58). In this
case, initial oxidation is performed by an alkane monooxygenase system
composed of a membrane-bound nonheme iron monooxygenase (a hydroxylase)
and two soluble proteins, rubredoxin and rubredoxin reductase, which
act as electron carriers between NADH and the hydroxylase
(40). The study of alkane hydroxylase diversity could
provide useful information about the mechanism of action and about the
regions involved in substrate specificity. Sequencing of small PCR
products from several alkane-degrading bacteria suggested that alkane
hydroxylases related to that of P. putida GPo1 are widely
distributed (49). However, only a few bacterial alkane
hydroxylases have been cloned and sequenced in addition to that of
strain GPo1, namely those of P. putida P1 (49),
Stenotrophomonas maltophilia (28), and some
Acinetobacter spp. strains (41, 49, 53).
Regulation of the expression of alkane degradation enzymes has received
much less attention. In the case of P. putida GPo1, the
expression of alkane degradation genes is controlled by the AlkS
protein (39), a transcriptional regulator that controls its own levels through a positive feedback mechanism (8).
Expression of the pathway is also modulated by catabolite repression,
depending on the carbon source being used (8, 20, 50, 62).
In the case of Acinetobacter sp. ADP1, expression of the
alkane hydroxylase gene is controlled by the transcriptional activator
AlkR, a protein unrelated to AlkS (42). In
Acinetobacter sp. strain M-1, which has two alkane
hydroxylases of different substrate specificities, two regulators have
been predicted (53). To our knowledge, regulation has not
been studied in other alkane degradation pathways.
The isolation and characterization of a number of bacterial strains
which can grow at the expense of residues obtained as end products of
crude oil processing has recently been described (63).
These residues show a considerable toxicity because they contain large
amounts of high-molecular-mass polyaromatic compounds and heavy
metals. The isolated strains did not degrade the polyaromatic compounds
but rather degraded the high-molecular-mass alkanes present in the
residue. We describe here the characterization of a gene coding for an
alkane hydroxylase in one of these strains, Burkholderia
cepacia RR10. Strain RR10 is interesting because it can grow at
the expense of alkanes containing 12 to at least 30 carbon atoms
(63), a range that is broader than that reported for most
bacterial isolates (43, 58). The alkane hydroxylase gene,
which was named alkB, is related to that of P. putida GPo1. Its promoter was identified, and its expression was
analyzed under different growth conditions. The regulation pattern
showed both differences and similarities to that of other alkane
hydroxylases characterized so far.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used are described in Table 1.
Plasmid pRR10 derives from pGEM7-Zf(+) by insertion between its
SacI and EcoRI restriction sites of a 550-bp DNA
fragment from B. cepacia RR10, which was obtained by PCR
using degenerated oligonucleotides designed to amplify conserved
regions of alkane hydroxylases related to the P. putida GPo1
AlkB protein, as described previously (49). Plasmids pALK7
and pALK8 were selected from a gene bank of B. cepacia RR10
total DNA in the cosmid vector pLAFR3 on the basis of their positive
hybridization to the 550-bp DNA fragment cloned into pRR10. They
contain overlapping DNA inserts of about 22 and 20 kbp, respectively.
Plasmid pALK12 was constructed by cloning a 3.4-kbp PstI DNA
segment from pALK8, hybridizing with the 550-bp probe, in the
PstI site of pUC18. This DNA fragment includes the 5' end of
the B. cepacia RR10 alkB gene. A 3.2-kbp BamHI fragment from plasmid pALK7, including the 3' end of
the B. cepacia alkB gene, was cloned into the
BamHI site of pUC18 to produce plasmid pALK14. To obtain a
transcriptional fusion of the promoter for the alkB gene to
the lacZ reporter gene, a 624-bp DNA fragment including
positions 
574 to +51 relative to the alkB transcription
start site (the alkB translation start is located at
position +117) was inserted between the EcoRI and BamHI sites of plasmid pUJ8. The fragment was obtained by
PCR using plasmid pALK12 as template and oligonucleotides
5'-aaggtcgaattcatgcaggc and 5'-ttttgtgcggatcctcgtcg
as primers, which include restriction sites for EcoRI or
BamHI. The resulting plasmid was named pALK31. Subsequently,
the 4.9-kbp NotI fragment with the
PalkB::lacZ fusion was cut from plasmid
pALK31 and inserted in the NotI site of
Mini-Tn5-Tc, obtaining plasmid pALK32. Plasmid pALK301
contains the complete B. cepacia alkB gene obtained by PCR
using total B. cepacia DNA as template; the DNA fragment
obtained was digested with EcoRI and cloned into the
EcoRI site of plasmid pNM185.
Media and culture conditions.
Cells were grown at 37°C in
Luria-Bertani (LB) medium or in minimal salts M9 medium
(45), the latter supplemented with trace elements
(4) and a carbon source (30 mM for organic acids or sugars
and 1% [vol/vol] for alkanes, alkanols, or fatty acids). To prepare
spent LB medium (46), B. cepacia RR10 was grown
in fresh LB to stationary phase; cells were eliminated by
centrifugation and subsequent filtration through a 0.45-µm-pore-size
filter, the pH was adjusted to 7.0, and the medium was filter
sterilized. Antibiotics were used at the following concentrations (in
µg/ml): ampicillin, 100; kanamycin, 50; tetracycline, 12;
streptomycin, 50.
Construction of a gene bank of B. cepacia
chromosomal DNA.
Total DNA from B. cepacia RR10,
obtained as previously described (3), was partially
digested with endonuclease Sau3AI and fractionated on a 10 to 40% linear sucrose gradient. DNA fragments 15 to 30 kbp in length
were cloned into the BamHI site of cosmid pLAFR3, using
Escherichia coli HB101 as the host for transfection. About
7,000 independent clones were obtained, which were pooled and stored at
70°C in LB medium containing 30% glycerol.
Recombinant DNA techniques.
General methods for DNA
manipulation and Southern blots were performed as described previously
(45). DNA was sequenced on both strands with an Applied
Biosystems DNA sequencer by the S.I.D.I-U.A.M. Sequencing
Service. The primers used to PCR amplify a 550-bp internal region of
the B. cepacia alkane hydroxylase as well as PCR conditions have been described previously (49). All other
amplifications were performed using standard protocols (annealing
temperature, 55 to 65°C; elongation temperature, 70°C; 30 cycles).
Plasmids were introduced into B. cepacia RR10 by conjugation
using plasmid pRK2013 as the donor of transfer functions in triparental
matings (13). In the case of Pseudomonas
fluorescens, plasmids were introduced by electroporation as
described previously (25).
Assay for -galactosidase.
An overnight culture of a
B. cepacia strain harboring a
PalkB::lacZ transcriptional fusion was
diluted to a final turbidity of about 0.04 either in LB medium (fresh
or spent, as indicated) or in minimal salts M9 medium supplemented with
the specified carbon source and in the absence or presence of the
indicated inducer. Cultures were grown at 37°C, and at different cell
densities aliquots were taken and -galactosidase activity was
measured as described by Miller (37). At least three
independent assays were performed in each case.
S1 nuclease analyses of mRNAs.
Total RNA was isolated from
bacterial cultures as described earlier (38). S1 nuclease
reactions were performed as described previously (3),
using 25 µg of total RNA and an excess of a 5'-end-labeled
single-stranded DNA (ssDNA) hybridizing to the 5' region of the mRNA.
The ssDNA probe was generated by linear PCR (62) using
plasmid pALK12 cut with endonuclease NotI as substrate. This
plasmid contains the 5' end of the B. cepacia RR10
alkB gene and about 2.3 kbp upstream from it; the
NotI target is located 213 nucleotides upstream of the RR10
PalkB translation start site.
Nucleotide sequence accession number.
The nucleotide
sequence of the 6,237-bp B. cepacia RR10 chromosomal region
containing the alkane hydroxylase gene was submitted to the EMBL data
bank under accession no. AJ293306.
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RESULTS AND DISCUSSION |
Identification of a gene coding for an alkane hydroxylase in
B. cepacia RR10.
A set of degenerated
oligonucleotides has been described which allows the PCR amplification
of a small internal region of genes related to the P. putida
GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases
(49). Using these oligonucleotides, a 550-bp DNA fragment
was obtained from B. cepacia RR10 coding for a polypeptide which shows 53% identity (71% similarity) to the corresponding internal region of the P. putida GPo1 alkane hydroxylase.
The amplified DNA fragment was used as a probe to screen a gene bank of
total B. cepacia RR10 DNA in cosmid pLAFR3. Two clones,
named pALK7 and pALK8, yielded a positive signal and contained
partially overlapping DNA inserts more than 20 kbp in length. A 3.4-kbp PstI DNA segment from pALK8 and a 3.2-kbp BamHI
DNA segment from pALK7, both hybridizing to the probe, were cloned in
pUC18, obtaining plasmids pALK12 and pALK14, respectively. The inserts
in these two plasmids were sequenced. They were found to contain
partially overlapping sequences spanning a 6,237-bp contig. A BLASTX
(1, 19) analysis of this sequence revealed the presence of
a 1,158-bp open reading frame (ORF) coding for a polypeptide showing
high similarity scores to known or predicted alkane hydroxylases. The highest scores were obtained for the putative alkane hydroxylase from
Mycobacterium tuberculosis H37Rv (54% identity), followed by the alkane hydroxylases from P. putida GPo1 (44%
identity), P. putida F1 (44% identity),
Acinetobacter sp. ADP1 (44% identity), and S. maltophilia (22% identity). A BLASTX search on the
Pseudomonas aeruginosa PAO1 genome
(http://www.pseudomonas.com) showed the presence of two putative alkane
hydroxylases with 39 and 41% identity, respectively, to the B. cepacia RR10 predicted alkane hydroxylase. A similar search at the
unfinished sequencing project of the Burkholderia pseudomallei genome (http://www.sanger.ac.uk) showed the
presence of a polypeptide with 80% identity to the B. cepacia RR10 protein. A phylogenetic tree showing the
relationships among these proteins is shown in Fig.
1. The B. cepacia RR10 alkane
hydroxylase clustered in a separate group together with the predicted
hydroxylases of M. tuberculosis and B. pseudomallei. The presence of the M. tuberculosis alkane hydroxylase in this group is surprising, considering that gram-positive bacteria are evolutionarily very distant from the Burkholderia group. The similarity between the different
alkane hydroxylases was distributed throughout the entire polypeptide (not shown), being particularly strong at a series of invariant histidine boxes which have been found to be important and highly conserved in integral membrane nonheme iron proteins such as
hydrocarbon hydroxylases and desaturases (26, 48, 49). The
size (386 amino acids) and molecular weight (43,953) of the polypeptide are also similar to those of known alkane hydroxylases. Therefore, the
cloned ORF most likely corresponds to an alkane hydroxylase and was
named alkB, following the nomenclature used for P. putida GPo1, the most thoroughly characterized alkane hydroxylase.
It is worth noting that among the reported PCR-amplified fragments of
genes related to P. putida GPo1 alkane hydroxylase obtained from several alkane-degrading bacteria (49), the B. cepacia RR10 alkB gene showed highest similarity to
that of B. cepacia ATCC 25416 (92.3% identity over the 182 amino acids in the PCR fragment).
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FIG. 1.
Similarity of the B. cepacia RR10 alkane
hydroxylase to other known or predicted bacterial alkane hydroxylases.
Proteins were aligned with ClustalW (55) at the European
Bioinformatics Institute web site (http://www2.ebi.ac.uk). The data
obtained were used to generate a phylogenetic tree with the
Phylodendron application at the IUBio Archive for Biology web site
(http://iubio.bio.indiana.edu). The identity of each alkane hydroxylase
to that of B. cepacia RR10 is indicated in parentheses.
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A BLASTX analysis of the regions sequenced upstream and downstream of
the B. cepacia RR10 alkB gene did not reveal the
presence of other ORFs showing similarity to genes known to play a role in alkane oxidation. A detailed description is presented in Fig. 2. An inverted repeat (15-bp stem, 4-bp
loop) followed by a run of T's, similar to a Rho-independent
terminator, was found 19 bp downstream from the stop codon of the
B. cepacia alkB gene, suggesting that transcription of
alkB is not coupled to that of any other ORF located
downstream of it. Altogether, these observations indicate that the gene
coding for the membrane component of the B. cepacia RR10
alkane hydroxylase is not clustered with other genes required for
alkane degradation. Present knowledge suggests that there is a large
variability in the clustering of alkane degradation genes in bacteria.
For example, the genes coding for alkane oxidation in the OCT plasmid
of P. putida GPo1 are grouped in two clusters
(58). In S. maltophilia a gene coding for a protein with similarity to rubredoxins but named rubredoxin reductase is located adjacent to that coding for the hydroxylase
(28), while the two analyzed Acinetobacter sp.
alkane hydroxylase genes are not linked to those encoding the
rubredoxin and rubredoxin reductases (17, 18, 41, 53).
Finally, the two putative alkane hydroxylases of P. aeruginosa PAO1 are located on separate sites of the chromosome
and are not clustered with other genes of the alkane degradation
pathway (unpublished observations).
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FIG. 2.
Analysis of the ORFs surrounding the B.
cepacia RR10 alkane hydroxylase gene. The 6,237-bp B.
cepacia DNA region sequenced was analyzed for the presence of
ORFs homologous to described genes of known function at the website
services of the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov) and of the European Bioinformatics
Institute (http://www2.ebi.ac.uk) using the program BLASTX
(19). The 1,158-bp ORF coding for a polypeptide showing
high similarity scores to known or predicted alkane hydroxylases was
named alkB (see the text). A polypeptide showing 30%
identity to the Methanococcus jannaschii hypothetical
protein MJ1207, which belongs to the acetyltransferase family of
proteins, was found upstream of alkB; it is indicated as
orf1. Downstream of orf1, an ORF was
present encoding a protein 43% identical to E. coli
MinC, followed by part of another ORF coding for a polypeptide
homologous to E. coli MinD (80% identity in the region
analyzed, which covers just the 112 N-terminal residues). E.
coli MinC and MinD are known to play an important role in the
process of cell division (reviewed in reference 31).
Downstream of alkB and oriented in the opposite
direction, an ORF was present encoding a protein 61% identical to the
E. coli seryl-tRNA synthetase (serS
gene). Upstream from serS and in the same orientation,
there is an ORF coding for a polypeptide homologous to part of an
E. coli hypothetical protein named YcaJ (84% identity
in the region analyzed, which covers only the C-terminal half of the
protein), the gene of which is also located upstream of
serS in E. coli. It is indicated as
orf2. The nucleotide sequence of the analyzed segment
was deposited at the EMBL data bank under accession no. AJ293306.
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Expression of the B. cepacia RR10
alkB gene in a heterologous host.
To show that the
cloned alkB gene indeed encodes a functional alkane
hydroxylase, the gene was transferred to P. fluorescens KOB2
1, a derivative of the alkane-degrading strain P. fluorescens CHAO in which part of the alkane hydroxylase gene has
been deleted (T. H. M. Smits, S. Balada, B. Witholt, and
J. B. van Beilen, unpublished data). For this purpose, the
B. cepacia RR10 alkB gene was cloned into the
broad-host-range expression vector pNM185 under the influence of the
Pm promoter. This promoter is activated by the
plasmid-encoded XylS activator in the presence of an appropriate inducer, such as 3-chlorobenzoate, a compound that is not used by
P. fluorescens KOB21 as a carbon source. Transfer of the
resulting plasmid, named pALK301, to P. fluorescens KOB21
restored the ability of this strain to grow on alkanes. Growth was
faster in the presence of the inducer 3-chlorobenzoate (doubling time
of about 40 h, compared to about 24 h for P. fluorescens CHAO), although it was also evident in its absence
(doubling time of about 70 h), probably because of a low basal
expression of the B. cepacia alkB gene in the absence of the
inducer. The recombinant strain-degraded alkanes have between 12 and 22 carbon atoms, a range similar to that observed for the parental strain
P. fluorescens CHAO in parallel control assays. All these
results show that the B. cepacia alkB gene encodes a
functional alkane hydroxylase.
Characterization of the promoter of the B. cepacia
RR10 alkane hydroxylase gene.
To analyze the expression of the
B. cepacia RR10 alkB gene in its original host,
total RNA was purified from B. cepacia RR10 cells grown in
minimal salts medium using tetradecane as the carbon source. The
transcription initiation site as well as the levels of expression at
different stages of the culture growth were determined by S1 nuclease
protection assays. As shown in Fig. 3, a
single transcription start site was detected, located 118 bp upstream of the alkB translation initiation codon. A clear 10
recognition sequence for the vegetative RNA polymerase (RNAP) was
present upstream of the start site (Fig. 3B). A moderately conserved
35 box for the vegetative RNAP could also be recognized 17 bp
upstream of the 5' end of the 10 box. The amount of transcripts
detected was reproducibly higher in cells collected at the start of the stationary phase than in cells collected in the exponential or in the
late stationary phase. This behavior, which has not been observed in
the few alkane degraders whose regulation has been studied to date, is
reminiscent of the E. coli promoters recognized by
S-RNAP (21, 22, 30). However, the
consensus for this form of RNAP in E. coli (5),
which is probably conserved in other gram-negative bacteria as well
(9), is not present in the PalkB promoter.
Alternatively, promoter activity could be under the control of a
quorum-sensing mechanism which is known to be present in B. cepacia (29). Despite several attempts, however,
ethyl-acetate extracts obtained from tetradecane-grown stationary-phase
cultures failed to stimulate expression of the PalkB
promoter in exponential phase. Addition to a fresh culture of up to
10% (vol/vol) of a spent M9 medium obtained from tetradecane-grown
cells did not have a positive effect either. The behavior of the
B. cepacia PalkB promoter could be explained by assuming
that it is regulated by a factor that is more active or that is present
at higher levels in stationary-phase cells. In fact, when the
PalkB::lacZ fusion was transferred to
E. coli CC118(
pir) or to P. putida KT2442, only low -galactosidase levels were observed (in the range of 150 to
200 Miller units), which were constant under all growth conditions. It
is likely, therefore, that this promoter requires a transcriptional
activator for induction.
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FIG. 3.
Identification of the promoter for the B.
cepacia alkane hydroxylase gene. (A) B. cepacia
RR10 was grown in either M9 minimal salts medium in the presence of
tetradecane (indicated as C14) or in LB medium in the
absence or presence of tetradecane. At different time points (early
exponential phase, eE; exponential phase, E; early stationary phase,
eS; or stationary phase, S), samples were collected and processed to
obtain total RNA. Transcripts originating upstream of
alkB were analyzed by S1 nuclease protection assays
using equal amounts of total RNA and an excess of ssDNA probe in all
cases. Samples were electrophoresed in parallel with a DNA size ladder
obtained by chemical sequencing (35) of the same ssDNA
used as a probe (lane M). The transcription start site is indicated by
an arrow. (B) Sequence of the promoter for the
alkB gene. The transcription start site observed
in the S1 nuclease protection assay is indicated with an arrow.
Positions at the 10 and 35 regions identical to those
recognized by the vegetative RNA polymerase at promoters in most
eubacteria (60) are highlighted in bold face. (C) The
growth curve of B. cepacia RR10 grown in M9 minimal
salts medium with tetradecane. Arrows indicate the time points
when samples were collected to obtain the total RNA used for the S1
nuclease protection assays described for panel A. Growth
was followed by measuring the increase in CFU on LB plates.
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To gain further insight into the regulation of the PalkB
promoter, its expression was investigated in cells of strain RR10 growing either in a rich LB medium or in a minimal salts medium using
citrate as a carbon source, in the absence or presence of tetradecane.
No expression of the promoter could be detected in LB medium
independent of the absence or presence of tetradecane and of the growth
phase (Fig. 3A). Similar results were found with cells grown in minimal
salts medium using citrate as a carbon source or using a mixture of
citrate and tetradecane (not shown). This indicates that expression of
alkB is regulated. Furthermore, these results suggest that
alkanes are not preferred carbon sources for B. cepacia, so
that when other more favorable carbon sources are used, expression of
the PalkB promoter is downregulated by a strong catabolite
repression. To further investigate this possibility, a transcriptional
fusion of promoter PalkB to the lacZ reporter gene was cloned into the suicide donor plasmid
pUT-mini-Tn5Tc and transferred to the B. cepacia
RR10 chromosome. A representative transconjugant, named CPCB2, was
selected for further analyses. Measurements of turbidity values in
cultures of B. cepacia RR10 growing on alkanes proved to be
unreliable due to the fine emulsification of the alkane, a problem
which impaired a confident determination of -galactosidase
activities. However, we observed that the
PalkB::lacZ fusion was efficiently
induced by tetradecanol, which is a good growth substrate for B. cepacia RR10 and does not present the emulsification problem.
Therefore, expression of the
PalkB::lacZ fusion was monitored by
measuring -galactosidase activity throughout the growth curve in
cells of strain CPCB2 grown in minimal salts medium containing
tetradecanol as a carbon source. As shown in Fig.
4, -galactosidase activity remained at
moderate levels (about 1,000 Miller units) during the exponential phase
of growth and rapidly increased as cells entered into stationary phase,
eventually reaching about sixfold higher values. Higher expression of
the PalkB promoter in stationary-phase tetradecanol-grown
cells agrees with the PalkB activity values observed in
tetradecane-grown cells with S1 nuclease protection assays (Fig. 3).
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FIG. 4.
Expression of the B. cepacia RR10
PalkB promoter in cells growing in a minimal salts
medium using tetradecanol as the carbon source. B.
cepacia strain CPBC2, a derivative of RR10 containing a
PalkB::lacZ transcriptional
fusion integrated into the chromosome, was grown in minimal salts
medium containing tetradecanol as the sole carbon source. Samples were
taken at different times, and the amount of -galactosidase present
in the cells was measured (represented as gray circles). Growth was
followed in parallel by counting CFU on solid media (dark
rectangles).
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Having established the behavior of the
PalkB::lacZ fusion, we proceeded to
test the effect of a number of carbon sources on the expression of the
PalkB promoter. Promoter activity in cells grown in LB
medium in the presence of tetradecanol was low and very similar to that
of cells grown in its absence (Table 2). In exponentially growing cells, -galactosidase levels in minimal salts cultures where tetradecanol was the sole source of carbon were
about 12-fold higher than those in LB cultures in the presence of
tetradecanol, a value that increased to 63-fold in stationary-phase cultures. These results agree with the repressing effect of the LB
medium observed by the S1 nuclease protection assays in cells growing
in the presence of tetradecane (Fig. 3A). A similar repressing effect
of the LB medium has been observed in the expression of the alkane
hydroxylase of P. putida GPo1, as well as in other catabolic
pathways for hydrocarbons (24, 33, 52), and is probably
due to the amino acids in LB (8, 62). However, the repression effect on the P. putida GPo1 pathway vanishes
when cells reach the stationary phase of growth. The behavior of the B. cepacia PalkB promoter was different. As shown in Table
2, stationary-phase cultures of strain CPBC2 grown in LB medium
containing tetradecanol as the inducer showed essentially the same
-galactosidase levels as exponential cultures. This agrees with the
transcription levels of the parental strain when grown in LB medium in
the presence of tetradecane (Fig. 3). Another interesting
characteristic of the catabolic repression effect of LB medium on the
promoters of the P. putida GPo1 alkane degradation pathway
is that repression is no longer observed when cells are grown on spent
LB medium (a medium that has already supported cell growth)
(62). Again, the behavior of the B. cepacia
PalkB promoter was different, since cells grown in a spent LB
medium did not show an increase in promoter activity in the presence of
an inducer (Table 2). This distinct behavior suggests mechanistic
differences in the way the metabolic status of the cell is coupled to
the expression of the alkane hydroxylase in P. putida GPo1
and in B. cepacia RR10.
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TABLE 2.
Expression of the B. cepacia PalkB promoter in
strain CPBC2 grown at the expense of different carbon sources
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The effects of several organic acids and sugars in cells cultured in
minimal salts medium containing tetradecanol were also analyzed. As
shown in Table 2, all the organic acids tested, as well as glucose,
fructose, lactose, and arabinose, strongly repressed expression of the
PalkB promoter in the presence of tetradecanol, both in the
exponential and stationary phases of growth. The effect of myristic
acid, the product of tetradecanol oxidation, was also analyzed. This
fatty acid decreased the induction effect of tetradecanol by about
4-fold in exponential phase and by about 16-fold in stationary phase.
This repression is interesting, since fatty acids are the product of
alkane oxidation. It is tempting to speculate that accumulation of
fatty acids may serve as a checkpoint to limit oxidation of alkanes
when cells sense that the -oxidation pathway is overloaded. In
summary, the behavior of the
PalkB::lacZ fusion is in full agreement
with the mRNA analyses performed with S1 nuclease protection assays.
Both assays indicate that expression of the PalkB promoter
is induced by hydrocarbons and that the induction is strongly repressed
by catabolite repression in the presence of other compounds that cells
probably metabolize preferentially.
Expression of the alkB gene in cells grown at the
expense of alkanes of different chain lengths.
Some
alkane-degrading bacterial strains are believed to contain only one
alkane hydroxylase, while others have two different (albeit related)
alkane hydroxylases. Examples of the former possibility are P. putida GPo1 (56, 58) and Acinetobacter sp.
ADP1 (41). P. aeruginosa PAO1 contains two
related alkane hydroxylases, although their individual contribution to
alkane metabolism has not been elucidated yet. Acinetobacter
sp. strain M-1 has two related alkane hydroxylases with different
substrate specificities; one of them preferentially oxidizes alkanes of
12 to 16 carbon atoms, while the other shows higher activity with
alkanes of more than 20 carbon atoms (32). It has been
shown that each hydroxylase is differentially regulated by a specific
transcription factor, each one responding to the presence of alkanes of
the chain length recognized by the corresponding hydroxylase
(53). To investigate the range of alkanes able to induce
the B. cepacia RR10 alkB gene, cells were grown
in minimal salts medium containing alkanes of either 12, 14, 16, 18, 20, 24, 26, or 30 carbon atoms as the sole carbon and energy source.
The expression of the PalkB promoter was analyzed in each
case by S1 nuclease protection assays. As shown in Fig. 5, all these alkanes induced the
PalkB promoter to similar levels. Unless we assume a
gratuitous induction by some of these alkanes, this suggests that the
hydroxylase may oxidize all these substrates. Since the inherent
difficulties in the genetic manipulation of B. cepacia RR10
has precluded us from obtaining a mutation in the alkB gene,
it is at present unclear whether B. cepacia RR10 contains
one or several alkane hydroxylases. However, several data are
compatible with the idea that the isolated alkB gene is the
only alkane hydroxylase gene present. First, the PCR amplification strategy to isolate the probe used for cloning, based on degenerated primers directed towards conserved regions of known alkane
hydroxylases, identified a single gene in several independent assays.
Second, the amplified DNA fragment afforded a single hybridization band in Southern blots performed with total B. cepacia RR10
chromosomal DNA (data not shown). Finally, the complementation analyses
described above showed that transfer of the B. cepacia alkB
gene to P. fluorescens KOB21 (lacking the alkane
hydroxylase that allows it to grow in C12 to
C18 alkanes) allowed it to grow at the expense of
alkanes having from 12 to 22 carbon atoms, a range similar to that
assimilated by the parental P. fluorescens CHAO strain in
parallel control tests. In this case, factors other than the substrate
range of the alkane hydroxylase possibly impede this strain to
assimilate longer alkanes (J. B. van Beilen, unpublished
observations).
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FIG. 5.
Expression of the B. cepacia RR10
alkB gene in cells growing at the expense of alkanes of
different chain lengths. B. cepacia RR10 was grown in
minimal salts medium containing either dodecane (C12),
tetradecane (C14), hexadecane (C16), octadecane
(C18), eicosane (C20), docosane
(C22), tetracosane (C24), hexacosane
(C26), or triacontane (C30) as the sole carbon
source. Samples were taken at the end of the exponential phase, and
expression of the PalkB promoter was analyzed as
described in the legend to Fig. 3. Lane M corresponds to the DNA size
ladder. The two panels correspond to gels containing equivalent amounts
of sample exposed for the same period of time.
|
|
Conclusions.
The work presented here shows that B. cepacia RR10 contains an alkane hydroxylase which is induced by
alkanes of very diverse chain lengths, its expression being fivefold
higher at the onset of the stationary phase than during exponential
growth. Transcription of the alkB gene was strongly
repressed by catabolite repression by many carbon sources that B. cepacia seems to prefer over alkanes. Many degradation pathways
for diverse hydrocarbons are controlled by catabolite repression
(reviewed in reference 10). However, the repression
exerted on the B. cepacia alkB gene is significantly stronger than that observed in other alkane degradation pathways. For
example, expression of the P. putida GPo1 alkane hydroxylase is also repressed by several organic acids, but the effect is much
milder (about fourfold [50, 62]). Similarly, citrate generates a strong repression in B. cepacia but has no
effect in strain GPo1. The repression observed in LB medium is also
different in the two strains. In P. putida GPo1, expression
of alkB is strongly repressed during exponential growth, but
repression ceases abruptly at the onset of the stationary phase
(62). Repression of the B. cepacia alkB gene in
LB medium was tightly maintained during the stationary phase.
Similarly, repression is not observed when P. putida GPo1 is
grown in a spent LB medium (62), although it is strong in
the case of B. cepacia. This may be due to metabolic and/or
mechanistic differences in the catabolic repression strategies of these
two bacterial species.
Hydrocarbons, and alkanes in particular, seem to be unfavorable growth
substrates for bacteria. This could be due at least in part to the fact
that many hydrocarbons show a certain level of toxicity for bacteria.
For example, despite providing abundant growth, alkane degradation is
known to impose a stress on cell physiology in P. putida
GPo1 (11, 12). Solvents like toluene activate multiple
responses in gram-negative bacteria (47), while methyl
benzoates switch on a stress response in E. coli and
probably in P. putida as well (34).
Alternatively (or in addition), the low solubility of
hydrocarbons and the high metabolic cost of biosurfactant production
limit their usefulness as carbon sources. Catabolite repression,
which is probably advantageous for bacterial fitness in their natural
environments, can pose limitations on the use of
hydrocarbon-degrading bacteria for diverse biotechnological
applications. A detailed knowledge of the global and specific
regulation mechanisms of bacterial pathways for hydrocarbons will
surely help to optimize their applications.
This work was supported by grants BIO97-0645-C02-01 and BIO2000-0939
from Comisión Interministerial de Ciencia y Tecnología to
F.R.
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Abstract
Introduction
Present address: IATE/P, EPFL, CH-1020 Lausanne, Switzerland.
