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Appl Environ Microbiol, April 1998, p. 1276-1282, Vol. 64, No. 4
Area of Microbiology, Department of Ecology,
Genetics and Microbiology, Faculty of Biology, University of
León, 24071 León, Spain,1 and
Departamento de Biologia, Universidad de Aveiro, 3800 Aveiro,
Portugal2
Received 3 June 1997/Accepted 17 January 1998
A 6.0-kb SalI DNA fragment containing an entire rRNA
operon (rrnB) was cloned from a cosmid gene bank of the
phytopathogenic strain Rhodococcus fascians D188. The
nucleotide sequence of the 6-kb fragment was determined and had the
organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without
tRNA-encoding genes in the spacer regions. The 5' and 3' ends of the
mature 16S, 23S, and 5S rRNAs were determined by alignment with the
rrn operons of Bacillus subtilis and other
gram-positive bacteria. Four copies of the rrn operons were
identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (=
NRRL B15096), also classified as R. fascians, produced five
rrn-hybridizing bands. An integrative vector containing a
2.5-kb DNA fragment internal to rrnB was constructed for
targeted integration of exogenous genes at the rrn loci.
Transformants carrying the exogenous chloramphenicol resistance gene
(cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium
and minimal medium and were fully stable for the integrated marker.
Rhodococcus fascians is a
gram-positive bacterium with a high G+C content belonging to the group
of lower actinomycetes (14) closely related to
corynebacteria. Strains of this species are of interest because they
are phytopathogenic (32), causing the formation of galls on
dicotyledonous plants (30) and malformations of bulbs of
monocotyledonous plants (24).
The molecular genetics of nonpathogenic corynebacteria have received
considerable attention (for reviews see references
23 and 29), but there are no
advanced recombinant DNA tools for studying molecular genetics of
plant-pathogenic bacteria such as R. fascians. Several
plasmids, including circular and high-molecular-weight linear plasmids,
are present in strains of R. fascians (7, 11).
Conjugative plasmids carrying genes determining resistance to cadmium
salts (10) or chloramphenicol (12) have been
characterized. One of these plasmids, pRF2, was used to develop
bifunctional vectors that also replicate in Escherichia coli
(12). By using these vectors, transformation of R. fascians strains has been obtained by electroporation (9,
11).
Some genes associated with phytopathogenicity were found in a 200-kb
linear plasmid in R. fascians D188 (7, 8).
Chromosomal genes also appear to be required to produce plant disease
(7). In order to clone and study additional genetic
determinants involved in plant pathogenicity, there is a need to
develop a system for chromosomal integration and expression of
homologous or heterologous DNA in well-characterized dispensable sites
of the R. fascians chromosome. As part of an effort in this
direction, we characterized the nucleotide sequence and organization of
an rRNA operon (rrnB) of R. fascians D188.
Although the 5S rRNA gene of this species (21) and the 16S
rRNA gene were amplified by PCR and used in phylogenetic analyses of
gram-positive bacteria previously (26), the complete
organization of the rrn operons and the number of rrn loci were not established.
Gene targeting is a useful strategy for introducing exogenous genes
into specific chromosomal regions. Due to their repetitive nature, rRNA
operons are very suitable targets for chromosomal integration of
foreign DNA fragments without modification of growth rates and
viability characteristics (5). In this paper we describe the
characterization of the rrnB locus and the use of
rRNA-encoding regions of R. fascians D188 as target sites
for integration and expression of the exogenous gene cmr, a
gene conferring chloramphenicol resistance (12).
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1. E. coli was grown at 37°C
in Luria broth or on Luria agar. When appropriate, ampicillin (50 µg/ml) was added to the medium. R. fascians was grown at
28°C in tryptic soy broth, on tryptic soy agar, or in minimal salts
thiamine medium (28). For selection of
chloramphenicol-resistant transformants of R. fascians the
antibiotic was used in the culture medium at a final concentration of
25 µg/ml.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of the rrnB Operon of
the Plant Pathogen Rhodococcus fascians and Targeted
Integrations of Exogenous Genes at rrn Loci
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Plasmid and total DNA purification. Small-scale preparations of E. coli plasmids were obtained as described by Birnboim and Doly (3). Recombinant cosmids were extracted by the alkaline lysis method, as described by Maniatis et al. (22). Total DNA was prepared from 100-ml overnight cultures. Cells were collected by centrifugation and resuspended in 5 ml of TES buffer (20 mM Tris-HCl [pH 8.0], 25 mM EDTA, 10.3% sucrose) and treated for 2 h at 37°C with lysozyme (0.25 mg/ml). Then, 1 volume of a solution containing 10% Sarkosyl and 0.5 M EDTA was added slowly. The lysate was treated with RNase (100 µg/ml) for 20 min at 37°C, followed by digestion with proteinase K (50 µg/ml) for 1 h at 50°C. After phenol extraction, DNA was precipitated from the aqueous phase with ethanol and resuspended in TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA).
Cosmid library construction.
Total DNA of R. fascians D188 was partially digested with Sau3AI. DNA
fragments with an average size of 40 to 50 kb were collected after
centrifugation in a 10 to 50% sucrose gradient, extracted with phenol,
and precipitated with ethanol. A 3-µg portion of this DNA was ligated
to a 10-fold molar excess of cosmid pHC79 (3 µg) (17) that
had been linearized with BamHI and treated with alkaline
phosphatase to prevent self-ligation. The ligation mixture was packaged
into 
phage particles in vitro by using a Gigapack II commercial kit
(Stratagene), and cosmid-containing phage particles were used to
transduce E. coli DH1 to ampicillin resistance (50 µg/ml),
which yielded 6 × 105 Apr transductants
per µg of R. fascians DNA. One thousand transductant colonies were transferred into 100 µl of Luria broth in 96-well microtiter plates and grown overnight at 37°C. Glycerol was added to
a final concentration of 20%, and the clones were stored at 
70°C.
DNA manipulations. Restriction enzymes, T4 DNA ligase, and calf intestinal phosphatase were obtained from Boehringer Mannheim and were used as recommended by the manufacturer. Restriction-generated fragments were separated in 0.7 to 1% agarose gels depending on their sizes and were isolated and purified with a Geneclean kit (BIO-101).
Electroporation of R. fascians. For electroporation, exponentially growing cells of R. fascians were concentrated 50-fold in a 30% polyethylene glycol 1000 solution (9). Electroporation was performed with a Gene-Pulser apparatus (Bio-Rad) by using the conditions described by Desomer et al. (9). E. coli was transformed by standard procedures (16).
DNA hybridizations. DNA fragments were transferred from agarose gels to nylon membranes (Amersham) by vacuum blotting. Probe labelling by the random priming method and hybridizations were performed with a digoxigenin kit from Boehringer Mannheim according to the manufacturer's instructions.
Sequence determination and analysis. The nucleotide sequence was determined on both strands with an automated dideoxy sequencing system (A.L.F. DNA sequencer; Pharmacia). The sequences used in this work for comparative studies were obtained from the EMBL and GenBank data banks. Analyses were performed with the MEGALIGN program (DNAStar Inc., Madison, Wis.).
Nucleotide sequence accession number. The nucleotide sequence of the rrnB operon of R. fascians D188 has been deposited in the EMBL and GenBank databases under accession no. Y11196.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Cloning of R. fascians rrn operons. A cosmid gene bank from R. fascians D188 was probed with a 2.7-kb HindIII DNA fragment of an rrn operon from Brevibacterium lactofermentum containing the entire sequence of the 16S rRNA gene and a small portion of the 23S rRNA gene (1). The rrn from Brevibacterium lactofermentum was used as a probe because a higher degree of similarity was expected between ribosomal DNA operons of R. fascians and Brevibacterium lactofermentum (both coryneform bacteria) than between operons of R. fascians and other gram-positive bacteria, such as Bacillus subtilis.
Ten positive hybridizing clones were identified by colony blotting. To identify small restriction fragments that gave positive hybridization, cosmid DNA was extracted and digested with several enzymes, and blots of the digests were hybridized with the same Brevibacterium lactofermentum rrn probe. By using this procedure a 6.0-kb SalI band and a 2.7-kb HindIII band were identified and subcloned in the E. coli vector pBluescript SK+. The resulting plasmids were designated pRS1 and pRH2, respectively. The 2.7-kb HindIII fragment was found to be internal to the 6.0-kb SalI fragment (Fig. 1A).
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Nucleotide sequence and organization of the ribosomal DNA locus. The 6.0-kb SalI insert of pRS1 was completely sequenced. In this fragment we found the previously reported R. fascians sequences encoding 16S and 5S rRNAs (accession no. X79186 and X15126, respectively). The presence of DNA encoding 23S rRNA was established by comparison with nucleotide sequences from Bacillus subtilis (accession no. K00637), E. coli (J01695), and Streptomyces lividans (M20148).
The rRNA genes in the 6.0-kb R. fascians DNA fragment were arranged in the order 16S rRNA-spacer-23S rRNA-spacer-5S rRNA (Fig. 1A). The spacer region between the 16S and 23S rRNA genes was 364 bp long, and the spacer region between the 23S and 5S rRNA genes was 134 bp long. No tRNA-like structures were identified in the spacer regions. The overall G+C content of the 6.0-kb fragment was 55.45 mol%, a value much lower than the average G+C content of the chromosomal DNA of R. fascians (64 mol%) (14). The G+C contents of the 16S rRNA-23S rRNA and 23S rRNA-5S rRNA spacers were 52.07 and 50.38 mol%, respectively. The 5' and 3' ends of the mature 16S rRNA were assigned to nucleotides 568 and 2088, respectively (Fig. 1B), on the basis of an alignment with the homologous gene of Bacillus subtilis (15). The 5' and 3' ends of the 5S rRNA gene were located at nucleotides 5733 and 5853, and the total length of the 5S rRNA was 121 nucleotides. Upstream from the 5' end of the 16S rRNA an RNase III cleavage site (AACUCAA) was found between nucleotides 377 and 383. From nucleotide 1444 to nucleotide 1456 the nucleotide signature for the high-G+C-content group (CUAAAACUCAAAG) is present. The base composition of the 3' end of the 16S rRNA is consistent with the presence of an anti-Shine-Dalgarno (SD) sequence. As shown in Fig. 1C, there is good complementarity between the 3' end of the 16S rRNA and regions located six to eight nucleotides upstream from the initiation codons AUG and GUG of several cloned R. fascians genes. The open reading frames analyzed correspond to the icl genes for isocitrate lyase (GenBank accession no. Z29367), a gene encoding a chloramphenicol resistance protein (Z12001), and a cluster of genes (ipt, P450, fdx) implicated in pathogenic traits (X62428). The number of bases involved in pairing ranged from four (fasR) to eight (ipt). The only exception was attE (accession no. Y09820), which encodes a protein of unknown function, is linked to the fasR gene, and did not show significant base pairing, suggesting that its ATG is not well-defined. The E. coli consensus Shine-Dalgarno sequence corresponded exactly to the SD sequence of the ipt gene. Alignment with the homologous 23S rRNA gene of Bacillus subtilis allowed us to assign the 5' and 3' ends of the R. fascians 23S rRNA gene to nucleotides 2452 and 5598, respectively (Fig. 1B). The total length of the R. fascians DNA encoding the 23S rRNA is 3,147 bp, which is slightly greater than the values reported for E. coli (2,904 bp) (4), Bacillus subtilis (2,927 bp) (15), and Streptomyces griseus (3,120 bp) (19), and the G+C content of this DNA is 54.97 mol%. Between positions 4009 and 4120 there was an insertion that included a 107-bp sequence that is found in several gram-positive bacteria (27). This insertion within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the gram-positive bacteria with high DNA G+C contents. This result indicates that R. fascians is phylogenetically related to corynebacteria and actinomycetes (27). As shown in Table 2, the 23S rRNA gene of R. fascians had a high percentage of nucleotides identical to the nucleotides in homologous genes of other gram-positive bacteria with high G+C contents, like Mycobacterium paratuberculosis (80.2% identical nucleotides), Mycobacterium leprae (74.5% identity), and Streptomyces species (75.2% identity to the Streptomyces lividans 23S rRNA gene and 75.1% identity to the Streptomyces griseus 23S rRNA gene). These values are higher than those obtained when comparisons were made with Bacillus subtilis 23S rRNA genes (49.6% identity) and E. coli 23S rRNA genes (53.2% identity), indicating that R. fascians is closely related to Mycobacterium species.
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Number of copies of rrn operons in R. fascians. The 6.0-kb SalI fragment sequenced does not contain BamHI sites. This restriction enzyme does not seem to have any target on the rRNA operons of R. fascians. Therefore, blots of BamHI-digested R. fascians D188 total DNA were hybridized with a 2.0-kb EcoRI-XbaI probe containing an internal fragment of pRH2. Four positive BamHI bands were obtained with the DNA of R. fascians D188 (Fig. 2, lane 7). The sizes estimated for these bands were 11.5, 10.4, 7.6, and 7.0 kb. It seems, therefore, that at least four copies of rRNA operons are present in the genome of this strain. To assess whether the copy number of the rRNA operons is the same in other isolates of this species, other strains classified as R. fascians (CECT 3001 and ATCC 12974) were examined in the same experiment. Strain ATCC 12974 also produced four BamHI bands, of 13.0, 11.4, 9.5, and 7.6 kb (Fig. 2, lane 6), whereas strain CECT 3001 produced five BamHI bands having estimated sizes of 15.0, 12.5, 10.8, 8.0, and 7.8 kb (Fig. 2, lane 5). R. fascians CECT 3001 also had a different genome size and produced a different restriction pattern with endonucleases that cut the genome infrequently (25), suggesting that this isolate is different from the R. fascians type strain (ATCC 12974). The pattern of hybridization of the SalI fragment with the 2.0-kb rrn probe was consistent with the pattern obtained with the BamHI fragments. Strain D188 produced four SalI bands (Fig. 2, lane 2), strain ATCC 12974 produced four bands (lane 3), and strain CECT 3001 produced five hybridizing bands (lane 4).
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Integrative vector targeted to rrn operons. The method used to construct an integrative vector used for targeted integrations at the rrn loci is shown in Fig. 3. The chloramphenicol resistance gene (cmr) from plasmid pRF30 (11) was subcloned in pBlueskript SK+ by digestion with XhoI and XbaI, which gave rise to 6.4-kb plasmid pSKCm. Ribosomal DNA fragments were obtained by digesting plasmid pRH2 with KpnI and XhoI. A 2.5-kb KpnI-XhoI fragment (containing parts of the 16S and 23S rRNA genes) was eluted from an agarose gel and ligated to pSKCm digested with the same enzymes. The resulting 8.9-kb plasmid, pIC44, cannot replicate in R. fascians (since it does not include an R. fascians replicon) but does replicate in E. coli. pIC44 contains the following two antibiotic resistance markers: ampicillin, expressed in E. coli cells; and chloramphenicol, expressed in R. fascians.
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Targeted integration at rrn loci of R. fascians. R. fascians D188.5, a nonpathogenic mutant derivative of D188 which lacks the pFiD188 linear plasmid and the pD188 circular plasmid (7), was electroporated with plasmid pIC44. After 7 days of incubation, 17 chloramphenicol-resistant colonies were isolated. The same experiment was repeated three times, and resistant colonies arose at a frequency of 20 to 50 transformants per µg of plasmid DNA.
Southern blots obtained with DNAs of several transformants were probed with a 2-kb EcoRI-XbaI fragment of pRH2 (Fig. 3) internal to the R. fascians rrnB operon. Typical results of this experiment are shown in Fig. 4. Lanes 1 through 4 show the results of four different integration events, each corresponding to changes in one of the hybridizing BamHI bands present in control wild-type strain D188 (Fig. 4, lane 5) and in its derivative, D188.5, the mutant used for electroporation (lane 6) (transformants 1 and 4 showed the same integration event). The hybridization patterns exhibited by the transformants are consistent with events involving chromosomal integration by single-recombination events in one of the rrn loci.
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Growth rates of mutants disrupted in an rrn operon. The growth curves of the transformed strains in rich media were similar to the growth curves of untransformed strain D188 (Fig. 5), indicating that integration of exogenous DNA sequences on a single ribosomal operon does not imply that there is a decrease in viability. However, in minimal salts thiamine medium there was a clear delay in the onset of the exponential growth rate, and the maximal cell density obtained was less than the maximal cell density in control cultures. Chloramphenicol resistance was maintained with total stability for at least 120 generations and during repeated transfers into media even when the transformed strains were cultivated in the absence of selective pressure.
|
) and mutant strain D188::pIC44.B
(
), which contains pIC44 integrated into the rrnB locus.
Data are the averages from four determinations at each time point in
duplicate experiments. O.D.590 nm, optical density at 590 nm.
Relevance of integrations at the rrn loci in the search for new pathogenicity traits. Our results indicate that it is possible to direct integrations introducing new sequences of DNA to a well-characterized chromosomal region by using the integrative vector targeted at the rrn loci. By using this integrative vector to transform nonpathogenic R. fascians mutants, it is possible to search for other genes that determine pathogenicity for plants. By integrating random DNA sequences from a pathogenic strain into the chromosome of a nonpathogenic strain, the effects of these genetic traits on phytopathogenicity can be observed. Work is now in progress to isolate and characterize pathogenicity traits by this approach.
The operons encoding rRNA contain usually redundant DNA (DNA that is present in several copies in the genomes of procaryotes). In the gram-positive bacteria, 6 operons have been found in Streptomyces coelicolor (31), 6 operons have been found in Staphylococcus aureus (33), 10 operons have been found in Bacillus subtilis (18), and 5 operons have been found in Corynebacterium glutamicum (2). Disruption of a single copy should not affect the viability and growth rates of a manipulated strain, as shown for E. coli (6, 13). Moreover, it has been reported that in E. coli deletion of four of seven ribosomal operons was required before any significant alteration of growth rate in minimal medium was observed (5). Our data for the growth rates of clones carrying a disrupted copy of the rrn operon clearly indicate that inactivation of one of the rrn loci does not greatly affect the growth rate. Therefore, it is unlikely that a disruption of rrn affects plant pathogenicity, although this question remains open.| |
ACKNOWLEDGMENTS |
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This work was supported by grant BIO92-0708 from the CICYT, Madrid, Spain. A.P. received a PFPI fellowship (Ministry of Education and Science, Madrid, Spain).
We thank J. Desomer and M. van Montagu for providing strains D188 and D188.5 and plasmid pRF30 and M. I. Corrales and R. Barrientos for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain. Phone: (34-87) 291505. Fax: (34-87) 291506. E-mail: degjmm{at}unileon.es.
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Top
Abstract
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Materials & Methods
Results & Discussion
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Appl Environ Microbiol, April 1998, p. 1276-1282, Vol. 64, No. 4
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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