Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci

Abstract

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.

FIG. 1

(A) Restriction map of a 6.0-kb R. fascians DNA fragment encoding the rrnB region. The 6.0-kb SalI and 2.7-kb HindIII fragments are indicated by arrows. The regions corresponding to the 16S, 23S, and 5S rRNAs are indicated by stippled boxes. (B) Alignment of the rrn gene of Bacillus subtilis with the corresponding regions of R. fascians encoding the 16S and 23S rRNAs. Note the strong conservation of the 5′ and 3′ regions. (C) Alignment of the 3′ end of the 16S rRNA gene of R. fascians with the regions upstream from the putative initiation codons of cloned genes from the same species. For each gene, pairing of at least four bases takes place. (D) Alignment of the first nucleotides of the R. fascians 16S rRNA-23S rRNA and 23S rRNA-5S rRNA spacers.

FIG. 2

Southern blot hybridizations of total DNAs of different strains of R. fascians with the 2.0-kb EcoRI-XbaI probe internal to rrnB. Lanes 1 and 8, DNA of recombinant cosmid p270; lanes 2 to 7, total DNA of R. fascians (lanes 2 and 7, strain D188; lanes 3 and 6, strain ATCC 12974; lanes 4 and 5, strain CECT 3001). The DNAs in lanes 1 to 4 were digested with SalI, and the DNAs in lanes 5 to 8 were digested with BamHI. Note that the DNA insert in cosmid pRF270 (used to isolate the sequenced rrn operon) corresponds to the second rrn band of BamHI-digested DNA of R. fascians D188 (arrow). The positions of size markers (λ digested with PstI plus λ digested with HindIII) are indicated on the right.

FIG. 3

Construction of integrative vector pIC44. The chloramphenicol resistance gene of plasmid pRF30 was introduced into Bluescript SK+, giving plasmid pSKCm. This plasmid was digested with KpnI and XhoI and ligated to a DNA fragment containing part of the 16S and 23S rRNA genes of the rrnB operon of R. fascians. Ap, ampicillin resistance gene; Cm, chloramphenicol resistance gene; MCS, multiple cloning site; ori, replication origin of the ColE1 plasmid.

FIG. 4

Southern hybridization of total DNAs of four different transformants (lanes 1 to 4), showing insertions of pIC44 in three of the four rrn operons compared to the hybridization patterns obtained for R. fascians D188 (lane 5) and D188.5 (lane 6), which were used as hosts in the transformation experiments. Lanes 1 and 4 show the same integration event. Blots were hybridized with a 2.0-kb EcoRI-XbaI probe internal to rrnB containing part of the 16S and 23S rRNA genes.

FIG. 5

Growth curves in complex medium (tryptic soy broth) (A) and in minimal salts thiamine medium (B) for wild-type strain R. fascians D188 (•) 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.