![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
J Bacteriol, June 1998, p. 3137-3143, Vol. 180, No. 12
Centro de Biología Molecular
"Severo Ochoa," UAM-CSIC, Universidad Autónoma de Madrid,
28049 Madrid, Spain
Received 16 December 1997/Accepted 29 March 1998
Despite the fact that the extreme thermophilic bacteria belonging
to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can
grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia
and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta,
1396:215-1997). We show here that this nar operon can be
transferred by conjugation to an aerobic Thermus strain,
enabling it to grow under anaerobic conditions. We show that this
transfer takes place through a DNase-insensitive mechanism which, as
for the Hfr (high frequency of recombination) derivatives of
Escherichia coli, can also mobilize other chromosomal
markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative
plasmid integrated into the chromosome. First, the nar
operon is absent from a plasmid-free derivative and from a closely
related strain. Second, we have identified an origin for autonomous
replication (oriV) overlapping the last gene of the
nar cluster. Finally, the mating time required for the
transfer of the nar operon is in good agreement with the
time expected if the transfer origin (oriT) were located
nearby and downstream of nar.
Most extreme thermophiles that live
in geothermal environments are strict anaerobes (3, 11) as a
consequence of the adaptation to the low solubility of oxygen at these
temperatures. However, members of the genus Thermus
constitute an exception to this general rule, being described
taxonomically as strictly aerobic chemorganotrophs (2).
However, we recently showed that one of the most thermophilic isolates
of this genus, Thermus thermophilus HB8, was able to grow
anaerobically when nitrate was present in the medium. Biochemical and
genetic evidence demonstrated that this ability was related to the
synthesis of a membrane-bound respiratory nitrate reductase complex
whose protein components, the We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains
HB8 and HB27 of T. thermophilus is the absence of plasmids
from the latter, the possibility that the nar operon could
be encoded by a transferable genetic element, such as a plasmid, was
considered.
In this article, we analyze this possibility and demonstrate that the
ability to grow by nitrate respiration can be transferred to the
aerobic strain T. thermophilus HB27 by conjugation. We also
relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus
HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal
genes in a time-dependent way.
Bacterial strains, phages, plasmids, and growth conditions.
T. thermophilus HB8 (ATCC 27634) and a Thermus
sp. strain (ATCC 27737) were obtained from the American Type Culture
Collection (Rockville, Md.). T. thermophilus HB27,
Thermus aquaticus YT1 (ATCC 25104), and T. thermophilus BPL7 were generously provided by Y. Koyama, M. Bothe,
and J. Fee, respectively. The mutant strains T. thermophilus
HB8 slrA::kat (9), T. thermophilus HB8 slpM::kat (9), and T. thermophilus HB8
narGH::kat (21) were used
for interrupted-mating experiments. E. coli JM109 [K-12
supE44
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Anaerobic Growth, a Property Horizontally
Transferred by an Hfr-Like Mechanism among Extreme
Thermophiles
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(NarG; 136 kDa), 
(NarH; 57 kDa),
and 
(NarI; 28 kDa) subunits, were homologous (about 48 to 50%
sequence identity) to those from mesophilic facultative anaerobes
(e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced
under low oxygen concentrations when nitrate was present
(21). In contrast to those described for most nitrate
reducers, the product of nitrate respiration was secreted to the growth
medium through an unknown transporter.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(lac-proAB) hsdR17 recA1 endA1
gyrA96 thi-1 relA1 F' (traD36 proAB+
lacIqZM15)] and TG1
[supE (nsdM-mcrB)5
(rK
McrB) thi
(lac-proAB) F' (traD36 proAB+
lacIqZM15)] were used for
cloning. Plasmids pUC119 (27) and pKT1 (15),
which contains a gene cassette encoding a thermostable kanamycin
nucleotidyltransferase (kat), were used for subcloning and
for the identification of oriV, respectively. Plasmid pMK18 (6) is a bifunctional E. coli-Thermus shuttle
vector used as a control in transformation experiments.
DNA isolation and labeling.
Standard methods were used to
purify, analyze, and manipulate DNA (22). Uniformly
32P-labeled DNA probes were obtained by use of random
hexanucleotide primers, [-32P]dCTP (3,000 Ci/mmol)
(Amersham Ibérica, Madrid, Spain), and the Klenow fragment of DNA
polymerase I. Essentially, 150 to 200 ng of DNA probe was denatured by
boiling and added to a 50-µl reaction mixture containing the
hexanucleotides (6 units of optical density at 260 nm
[OD260]/ml), dATP, dGTP, and dTTP (0.1 mM each), -mercaptoethanol (6 mM), MgCl2 (20 mM), and bovine serum
albumin (0.4 mg/ml) in 50 mM Tris-HCl-200 mM HEPES buffer (pH 7). The Klenow fragment (2 U) and 25 mCi of [-32P]dCTP (3,000 Ci/mmol) were added to this mixture, and the labeling reaction was
developed at 30°C for 10 h. After this period, unincorporated nucleotides were removed by chromatography in a Sephadex G-50 column.
The radiactive probes used were as follows: probe A is a 1.2-kbp
BamHI fragment containing 3' and 5' regions of
narG and narH, respectively (21), and
probe B is a 3.5-kbp KpnI/SalI fragment from
plasmid pNIT9kat (this work).
PFGE. After T. thermophilus cells were placed in 1% (wt/vol) agarose, intact DNA was obtained by the method described by Marin et al. (17). Plasmids were removed from these agarose plugs by pulsed-field gel electrophoresis (PFGE) for 36 h at 170 V/cm and a pulsing time of 150 s in 1% (wt/vol) agarose (SeaKem LE agarose; FMC) in TBE buffer (100 mM Tris, 100 mM boric acid, 0.2 mM EDTA [pH 8]) by use of a contour-clamped homogeneous electric field system (Pulsaphor apparatus; LKB) (4). Plugs containing only chromosomal DNA were then treated in situ with restriction enzyme NdeI (New England BioLabs) as described previously (17, 24), and the fragments obtained were subsequently separated by PFGE for 36 h at a field strength of 10 V/cm and a pulsing time of 40 s. After ethidium bromide staining, the sizes of the DNA fragments were calculated by comparison to lambda phage concatemers (50 kbp per monomer).
Southern blot analysis.
Essentially we followed the protocol
described by Sambrook et al. (22). DNA fragments separated
by agarose gel electrophoresis were capillary transferred for 16 h
to a nylon membrane (Hybond N; Amersham) in 20× SSC buffer (1× SSC is
0.15 M NaCl plus 20 mM sodium citrate). After UV cross-linking,
hybridization was carried out for 16 h at 42°C with ~2 × 106 cpm of an appropriate 32P-labeled DNA probe
(see above) in 10 ml of hybridization solution (6× SSC, 0.1% sodium
dodecyl sulfate [SDS], 100 mg of denatured salmon sperm DNA per ml).
Finally, nonspecifically bound probe was removed by washing in 2×
SSC-0.1% SDS at 62°C, and the remaining radioactivity was detected
by autoradiography at 
70°C.
Construction of pNITkat plasmids and transformation. The kat gene, encoding the thermostable kanamycin nucleotidyltransferase, was isolated from plasmid pKT1 and inserted into the PstI restriction site of the narI gene (21) to obtain plasmid pNIT9kat. From this plasmid, a 3.5-kbp KpnI/HindIII DNA fragment containing regions downstream of nar was cloned into the corresponding restriction sites of pUC119 to obtain plasmid pNIT5. Then, plasmids pNIT5kat1 and pNIT5kat2 were obtained by partial digestions with SmaI, followed by replacement with the kat gene cassette. Later, plasmids pNIT5kat1XSI and pNIT10kat were obtained from pNIT5kat2 by deletion of 0.6-kbp XhoI/SalI and 1.4-kbp EcoRI/HindIII DNA fragments, respectively.
Natural competence in T. thermophilus was induced by growing cells at 70°C with aeration to an OD550 of 0.5 in TB containing 1 mM MgCl2 and 0.5 mM CaCl2. Then, 0.5-ml aliquots of the culture were made, and plasmid DNA (1 µg) was immediately added. After 2 h of incubation, cells were plated on kanamycin (30 µg/ml)-containing plates and further incubated for 24 to 48 h at 70°C to obtain colonies. E. coli cells were made competent and transformed as described previously (5, 19).DNA sequencing and computer analysis.
Plasmid DNA was
sequenced by the dideoxy chain termination method (23). For
the manual method, we used 7-deaza-dGTP, modified T7 DNA polymerase
(Sequenase 2.0; U.S. Biochemicals), and [-35S]dATP
(1,000 Ci/mmol) (Amersham). For the automated method, sequences were
obtained with an Applied Biosystems sequencer. Universal primers for
M13mp and pUC vectors and oligonucleotides synthesized from partial
sequences (Isogen Bioscience, Maarshen, Holland) were used for priming.
Partial sequences were overlapped and analyzed with Wisconsin Genetics
Computer Group software (7). Both strands of the template
DNA were completely sequenced.
Horizontal transfer of the nar operon. A chloramphenicol-resistant strain (T. thermophilus HB27Camr) was isolated directly by plating 108 T. thermophilus HB27 cells on plates containing chloramphenicol (20 µg/ml). For mating experiments, the donor (T. thermophilus HB8) and recipient (T. thermophilus HB27Camr) strains were mixed in a 100:1 (receptor/donor) ratio in 100 ml of TB medium containing KNO3 (20 mM) and incubated for 8 h at 70°C with low-speed stirring (100 rpm). A 500-µl sample of the culture was then inoculated into 100 ml of medium containing KNO3 (20 mM) and chloramphenicol (20 µg/ml) and incubated overnight at 70°C without stirring to allow the selection of exconjugants. Finally, cells from this culture were inoculated into capped test tubes containing 10 ml of nitrate medium overlaid with mineral oil up to the screw cap and incubated for 24 h at 70°C. The latter process was repeated five times to guarantee the isolation of an anaerobic culture. Parallel negative controls in which both donor and recipient strains were incubated separately and subjected to the same selective procedure were developed.
For interrupted mating, donors (different T. thermophilus HB8::kat derivatives) and recipients (T. thermophilus HB27Camr) were mixed as described above in a medium containing 6 mM MgCl2 and DNase I (100 µg/ml) to exclude the possibility of natural competence-mediated transfer. Aliquots (300 µl) of the mixtures were incubated at 70°C for different times before being vortexed vigorously. Then, 200 µl from each tube was added to 300 µl of prewarmed medium containing chloramphenicol (30 µg/ml) and incubated for 3 h at 70°C under strong aeration. Finally, 200 µl of this mixture was spread on kanamycin (30 µg/ml) and chloramphenicol (20 µg/ml) agar plates and incubated for 48 h at 70°C.SDS-PAGE and Western immunoblotting. Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (14). For Western blotting, an antiserum raised against a C-terminal fragment of NarG was used (21). Specifically bound antibodies were detected with the enhanced chemiluminescence Western blotting analysis system from Amersham International.
Nitrate reductase activity. The nitrate reductase activity of triplicate cell samples corresponding to an OD550 of 0.6 was measured after permeabilization with tetradecyltrimethylammonium (20% [wt/vol]) with methyl viologen as the electron donor (21, 25). One enzyme unit under these conditions was defined as the amount that produced 1 nmol of nitrite per min at 80°C.
Nucleotide sequence accession number. Accession number AJ225043 has been assigned by the EMBL gene bank to the nucleotide sequence of the oriV region that we studied (see Fig. 6).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The nar operon is absent from most Thermus strains. In order to check if the nitrate respiration ability shown by T. thermophilus HB8 was present in other strains of this genus, we inoculated the isolates Thermus sp. strain ATCC 27737, T. aquaticus YT1, T. thermophilus HB27, and T. thermophilus BPL7 and the nar mutant T. thermophilus HB8 narGH::kat (21) into TB medium containing nitrate and incubated them for 48 h at 70°C under anaerobic conditions. The results of such experiments demonstrated that none of these strains was able to grow under these conditions, while the control strain T. thermophilus HB8 grew to an OD550 of 0.8 (data not shown).
To check whether these results were related to the absence of the nar operon or were due to problems in its expression, we developed a parallel Southern blot of BamHI-digested total DNA from these strains by using 32P-probe A as a marker for the presence of the nar operon. As shown in Fig. 1, a 1.2-kbp labeled fragment detected in T. thermophilus HB8 (lane 1) was absent from all the other strains analyzed. As a negative control, we included the narGH::kat strain (Fig. 1, lane 2), an insertional derivative of T. thermophilus HB8 from which the fragment used as a probe was deleted (21).
|
The nar operon is horizontally transferred. The results reported above, especially the absence of the nar operon from T. thermophilus BPL7, a plasmid-free derivative of T. thermophilus HB8 (18), suggested the likely location of the nar operon to be in a plasmid and prompted us to test its putative transfer to other aerobic Thermus strains.
To test this possibility in conjugation experiments, a chloramphenicol-resistant derivative of T. thermophilus HB27 (T. thermophilus HB27Camr) was used as a recipient and T. thermophilus HB8 was used as a donor (see Materials and Methods). After the 8-h mating period, nitrate-respiring and chloramphenicol-resistant strains were isolated by growing the cells consecutively under microaerophilic and completely anaerobic conditions. Parallel controls in the absence of the recipient were developed to check the possible selection of chloramphenicol-resistant derivatives of strain HB8 during this process. As shown in Fig. 2A, the chloramphenicol-resistant organism selected by this experiment (hereafter referred to as T. thermophilus HB27Camr::nar) was able to grow under anaerobic conditions with nitrate, while the parental recipient organism, T. thermophilus HB27Camr, was not.
|
Expression of the nar operon in the exconjugant
T. thermophilus
HB27Camr::nar.
As shown in Fig.
3A, SDS-PAGE protein patterns of T. thermophilus HB8 (donor, lanes 2 to 4) were easily distinguishable
from those of T. thermophilus HB27Camr
(recipient, lane 5) and T. thermophilus
HB27Camr::nar (exconjugant, lanes 6 and 7). In fact, the protein profiles of the recipient (Fig. 3A, lane
5) and the exconjugant (lanes 6 and 7) were almost identical, thus
confirming their genetic relationship. However, a 140-kDa protein
specifically detected in the exconjugant (Fig. 3A, lanes 6 and 7) was
absent from the parental recipient (lane 5) but apparently present in
T. thermophilus HB8 cells grown under microaerophilic (lane
3) and anaerobic (lane 4) conditions. A parallel Western blot (Fig. 3B)
clearly identified this 140-kDa protein as NarG, the subunit of the
thermophilic nitrate reductase (21).
|
Transfer of the nar operon occurs through an Hfr-like mechanism. The results of the experiments shown in Fig. 2 and 3 suggested that the nar operon was transferred from the donor chromosome to that of the recipient to obtain the exconjugant T. thermophilus HB27Camr::nar. Since we could not detect the presence of DNA in the conjugation media (as would be expected from the use of a bacteriostatic antibiotic as a selective criterion), the mechanism most likely responsible for this transfer was conjugation. However, the natural competence capability described for Thermus spp. (12) still remained a less likely explanation.
To distinguish between these two possibilities, we used a classic interrupted-mating experiment (see Materials and Methods) to check whether there was a time dependence for the transfer of different genes. If the transfer were ordered, a conjugative mechanism could be inferred. In contrast, random transfer would indicate transformation as the most likely mechanism. For these experiments and to make the selection easier (kanamycin resistance), we used as donors different T. thermophilus HB8::kat mutants, including the narGH::kat derivative (9, 21). As shown in Table 1, transfer of the slpM::kat mutation required less than 10 min, followed shortly afterward by the slrA::kat mutation. Nevertheless, about 50 min of mating was required for transfer when the narGH::kat mutant was used as a donor. Thus, we concluded that there was a time dependence for the transfer of each mutation assayed.
|
Identification of a replicative origin (oriV) downstream of the nar operon. During the construction of narI::kat insertion mutants of T. thermophilus HB8 (unpublished data), we detected a high transformation efficiency with the circular form of plasmid pNIT9kat, which contains part of the nar operon and its downstream region (Fig. 4). In fact, the transformation efficiency observed (Table 2) was similar to that obtained with the bifunctional E. coli-Thermus shuttle vector pMK18 (6). Therefore, this result suggested the possible presence downstream of the nar operon of sequences encoding an origin for autonomous replication (oriV) in Thermus strains.
|
|
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Two important conclusions arise from the results presented in this article. First, we describe for the first time for strains of the genus Thermus a conjugative mechanism which is also able to mobilize chromosomal genes in a time-dependent manner. The second conclusion is more general, since we show that the ability to grow under anaerobic conditions may be encoded within a mobilizable element, thus making doubtful the relevance of the "aerobic" character of an organism in formal taxonomy (2).
The transfer of the nar operon from the facultative anaerobe T. thermophilus HB8 (donor) to the aerobe T. thermophilus HB27Camr (recipient) resulted in the isolation of a Camr facultative anaerobe. The nature of this organism as a derivative of the recipient was assessed in several ways. First, parallel controls in the absence of the recipient did not allow the isolation of any Camr mutant from the donor. Second, the total protein pattern of the organism selected after the conjugation was identical to that of the recipient and clearly different from that of the donor (compare lanes 5 and 6 with lanes 2 and 3 of Fig. 3). Finally, the profiles of NdeI digestion fragments of chromosomal DNA from the recipient and the exconjugant were identical except for the insertion of a 30-kbp DNA fragment.
A second point concerns the mechanism of transfer. That the transfer of the nar operon was due to conjugation and not to the natural competence described for Thermus was supported by different arguments. (i) The existence of external DNA during the mating period was unlikely due to the use of a bacteriostatic selective criterion (chloramphenicol) in the expression medium. In fact, we did not detect DNA in the medium after the conjugation. (ii) The transfer also took place in the presence of DNase I, an enzyme which was still active at 70°C even after the mating period. (iii) Different mating times were required to transfer different genes, a situation that would not occur if a transformation phenomenon were implicated.
The time-dependent transfer of chromosomal genes could be explained only on the basis of a mechanism similar to that of the Hfr strains of E. coli. In these strains, a plasmid (F) inserted into the chromosome provides the genes (tra) and an origin (oriT) required to drive the transfer of a single-strand copy of the whole chromosome to a recipient cell (16). As oriT is located within the plasmid sequence, plasmid-encoded genes located upstream of oriT are the last to be transferred, requiring about 100 min of mating in the E. coli system. Keeping in mind that the circular chromosome of T. thermophilus HB8 is about 1.740 kbp long (1) and assuming a rate of transfer similar to that in E. coli (chromosome size about 4.200 kbp), the 45 to 50 min of mating required to transfer the narGH::kat mutation could be expected if oriT were located immediately downstream of the nar operon. Unfortunately, the limited repertoire of gene markers makes impossible at the present time a detailed genetic analysis to check the putative association between the nar operon and oriT.
Additional evidence which supported the association between the nar operon and a conjugative plasmid was the fortuitous identification of a replicative oriV downstream of the nar operon (Fig. 4) which was functional in T. thermophilus HB8 and in T. thermophilus HB27Camr::nar but not in T. thermophilus HB27Camr or T. thermophilus BPL7 (Table 2). This fact suggests the existence of a gene carried by the transferred fragment, whose expression is required for the replication of this oriV. It is possible that the role of this factor is the identification of specific sequences at oriV, allowing the melting of DNA and the subsequent recruiting of a replication complex, as is the situation with the Rep protein(s) from many other plasmids (13).
The sequence of the region containing oriV revealed the presence of inverted and direct repeats downstream of a sequence which encodes the C-terminal part of a protein homologous to the nitrite extrusion protein NarK (Fig. 6). Although the presence of downstream T-rich sequences suggests a role for ir2 as a Rho-independent transcription terminator, the other repeats found may be related to the binding of the putative Rep factor mentioned above. Nevertheless, their role in replication and/or transcription is not known at present, and further deletion analysis is required to determine their role.
The above results support the existence of a conjugative plasmid which has been integrated into the chromosome of T. thermophilus HB8. Two plasmids, pTT8 and pVV8, have been described for T. thermophilus HB8 (26). Of these, only pVV8 is a likely candidate to be conjugative because of its larger size (26), its ability to confer an aggregation phenotype, and its ability to integrate into the chromosome through homologous regions (18). However, Southern blot assays revealed that neither pVV8 nor pTT8 hybridized with a nar probe (data not shown). Consequently, the oriV that we identified downstream of the nar operon belongs to a different plasmid. In this sense, its absence from T. thermophilus BPL7, a derivative of HB8, means that the nar-carrying plasmid was lost during the complex procedure followed for its selection, which included long-term growth under 100% oxygen, nitrosoguanidine mutagenesis, and ampicillin enrichment (18). Such a complex selection could have induced excision from the chromosome and the loss of the nar-carrying integrated plasmid to yield T. thermophilus BPL7.
After its transfer to the recipient, the expression of the nar operon in the exconjugant T. thermophilus HB27Camr::nar was still regulated by nitrate and oxygen. Whether this result was due to the simultaneous transfer of both the oxygen and the nitrate sensors or to their previous presence in the recipient cannot be answered at present. However, the inability of the recipient to use nitrate (21) makes the last possibility most unlikely, at least for the nitrate sensor system.
It is noteworthy that nar expression was even more gradual in the exconjugant than in T. thermophilus HB8; although full induction in HB8 was reached under microaerophilic conditions, completely anoxic conditions were required for full expression in the exconjugant. Furthermore, in spite of the expression of similar amounts of NarG protein (Fig. 3, lanes 4 and 7, for the HB8 and HB27Camr::nar strains, respectively), the enzymatic activity in the exconjugant strain was three times lower than that in strain HB8, suggesting that part of the enzyme synthesized was inactive. Accordingly, the exconjugant strain yielded less growth under anaerobic conditions than did the parental donor strain (Fig. 2A).
All of these data support the fact that the ability of T. thermophilus HB8 to respire nitrate is encoded in a genetic element which can be transferred to aerobic strains of the same genus, changing an obligate character to facultative. In fact, the unexpected presence of nar operons in certain strains of supposedly obligate aerobes such as Pseudomonas fluorescens (20) and Bacillus subtilis (10) could be due to a process of horizontal transfer similar to that described here. If this were the case, the requirement for oxygen, commonly used in formal taxonomy as one of the main characteristics for the classification of microorganisms, could be viewed as meaningless.
| |
ACKNOWLEDGMENTS |
|---|
The technical assistance of J. de la Rosa is greatly appreciated.
This work was supported by project BIO97-0665 from the Comisión Interministerial de Ciencia y Tecnología (C.I.C.Y.T.) and by an institutional grant from Fundación Ramón Areces. S. Ramírez-Arcos holds a fellowship from Instituto de Cooperación Iberoamericana (I.C.I.).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa," UAM-CSIC, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Phone: 34-91-3978099. Fax: 34-91-3978087. E-mail: JBERENGUER{at}trasto.cbm.uam.es.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. |
Borges, K. M., and P. L. Berquist.
1993.
Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.
J. Bacteriol.
175:103-110 |
| 2. | Brock, T. D. 1984. Genus Thermus Brock and Freeze 1969, 295AL, p. 333. In N. R. Krieg, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, Md. |
| 3. | Burggraf, S., G. J. Olsen, K. O. Stetter, and C. R. Woese. 1992. A phylogenetic analysis of Aquifex pyrophilus. Arch. Microbiol. 15:352-356. |
| 4. | Chu, G., D. Vollrath, and R. W. Davis. 1984. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234:1582-1585. |
| 5. | Dagert, M., and S. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23-28[Medline]. |
| 6. | DeGrado, M. 1996. In Caracterización de orígenes de replicación autónoma del género Thermus. Ph.D. thesis. Universidad Autónoma de Madrid, Madrid, Spain. |
| 7. | Devereaux, J., P. Haeverli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. |
| 8. |
Fernández-Herrero, L. A.,
G. Olabarría,
J. R. Castón,
I. Lasa, and J. Berenguer.
1995.
Horizontal transfer of S-layer genes within Thermus thermophilus.
J. Bacteriol.
177:5460-5466 |
| 9. | Fernández-Herrero, L. A., G. Olabarría, and J. Berenguer. 1997. Surface proteins and a novel transcription factor regulate the expression of the S-layer gene in Thermus thermophilus HB8. Mol. Microbiol. 24:61-72[Medline]. |
| 10. | Hoffmann, T., B. Troup, A. Szabo, C. Hungerer, and D. Jahn. 1995. The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. FEMS Microbiol. Lett. 131:219-225[Medline]. |
| 11. | Huber, R., R. A. Langworthy, H. Köning, M. Thomm, C. R. Woese, U. B. Sleyter, and K. O. Stetter. 1986. Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacterium growing up to 90°C. Arch. Microbiol. 144:324-333. |
| 12. |
Koyama, Y.,
T. Hoshino,
N. Tomizuka, and K. Furukawa.
1986.
Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.
J. Bacteriol.
166:338-340 |
| 13. |
Kües, U., and U. Stahl.
1989.
Replication of plasmids in gram-negative bacteria.
Microbiol. Rev.
53:491-516 |
| 14. | Laemmli, U., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:575-599[Medline]. |
| 15. | Lasa, I., J. R. Castón, L. A. Fernandez-Herrero, M. A. Pedro, and J. Berenguer. 1992. Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus. Mol. Microbiol. 11:1555-1564. |
| 16. | Low, K. B. 1996. Hfr strains of Escherichia coli K-12, p. 2402-2405. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. |
| 17. | Marín, I., R. Amils, and J. P. Abad. 1995. Linear extrachromosomal DNA of Thiobacillus cuprinus DSM 5495 in relation to its chemolithotrophic growth. FEMS Microbiol. Lett. 134:75-78. |
| 18. | Mather, M., and J. Fee. 1990. Plasmid-associated aggregation in Thermus thermophilus HB8. Plasmid 24:45-56[Medline]. |
| 19. | Miller, J. H. 1992. In A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 20. | Philippot, L., A. Clays-Josserand, R. Lensi, I. Trinsoutreau, P. Normand, and P. Potier. 1997. Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and the cloning and sequencing of its corresponding genes. Biochim. Biophys. Acta 1350:272-276[Medline]. |
| 21. | Ramírez-Arcos, S., L. A. Fernández-Herrero, and J. Berenguer. 1997. A thermophilic nitrate reductase is responsible for the strain specific anaerobic growth of Thermus thermophilus HB8. Biochim. Biophys. Acta 1396:215-227. |
| 22. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. In Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 23. |
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467 |
| 24. | Smith, C. L., S. R. Kolco, and C. R. Cantor. 1988. Pulse field electrophoresis and the technology of large DNA molecules, p. 41-72. In K. Davies (ed.), Genome analysis: a practical approach. IRL Press, Oxford, England. |
| 25. | Snell, F. D., and C. T. Snell. 1949. In Colorimetric methods of analysis. Van Nostrand, New York, N.Y. |
| 26. | Vasquez, C., J. Villanueva, and R. Vicuña. 1983. Plasmid curing in Thermus thermophilus and Thermus flavus. FEBS Lett. 158:339-342. |
| 27. | Vieira, J., and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3-11[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
and 
29 phages were used as size markers (lane M).
