Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

Abstract

Riemerella anatipestifer is a member of the family Flavobacteriaceae and a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whether R. anatipestifer is also competent for natural transformation has not been investigated. Here, we showed that R. anatipestifer strain ATCC 11845 can uptake the chromosomal DNA of R. anatipestifer strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for R. anatipestifer ATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10^-5 transformants. Competition assay experiments showed that R. anatipestifer ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as Escherichia coli DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10^-9 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing R. anatipestifer DNA fragments (transformation frequencies of ∼10^-7 transformants). Finally, we found that the R. anatipestifer RA-CH-1 strain was also naturally transformable, suggesting that natural competence is widely applicable for this species. The findings described here provide important tools for the genetic manipulation of R. anatipestiferIMPORTANCERiemerella anatipestifer is an important duck pathogen that belongs to the family Flavobacteriaceae At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of R. anatipestifer remain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not in R. anatipestifer For the first time, we showed that natural transformation occurred in R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1. Then, we established an easy gene knockout method in R. anatipestifer based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in R. anatipestifer.

Keywords: Riemerella anatipestifer; natural transformation; targeted mutagenesis.

FIG 1

Determination of erythromycin resistance gene was inserted in the genome of R. anatipestifer ATCC 11845. The genomes of six resistance clones were extracted and used as the templates to perform PCR using primers Erm P1 and Erm P2 (see Table S2 in the supplemental material). Lanes 1 to 6, clone 1 to clone 6; +, positive control (the genome of RA-CH-1); −, negative control (the genome of R. anatipestifer ATCC 11845); and M, DNA ladder (Biomed, Beijing, China).

FIG 2

Verification of mutant strain R. anatipestifer ATCCΔRA0C_1193. Lane M, MD103DNA marker (Biomed, Beijing, China). The 16S rRNAs were amplified from R. anatipestifer wild-type strain R. anatipestifer ATCC and R. anatipestifer ATCCΔRA0C_1193 using primers 16S rRNA P1 and 16S rRNA P2 (lane 1); the RA0C_1193 gene was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers RA0C_1193 P1 and RA0C_1193 P2 (lane 2); the Erm^r cassette was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers Erm^r P1 and Erm^r P2 (lane 3); the left flanking sequence of RA0C_1193 and the Erm^r cassette was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers RA0C_1193up P1 and Erm^r P2 (lane 4); the Erm^r cassette and right flanking sequence of RA0C_1193 was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers Erm^r P1 and RA0C_1193down P2 (lane 5).

FIG 3

Effect of donor DNA amounts on transformation efficiency. Donor DNA (0. 1 to 4,000 ng) was added to competent bacterial cultures and incubated for 1 h. Erm^r transformants were selected. The number of transformants increased as the donor DNA concentration increased, and the saturation level of transforming DNA was approximately 1 μg.

FIG 4

Dependence of the transformation efficiency on the incubation time with the donor DNA. The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic PCR fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm^r transformants were selected after 1 h of incubation.

FIG 5

Transformation competition experiments. Competent R. anatipestifer ATCC 11845 was transformed with 1 μg RA0C_1193 mutagenic PCR fragments alone (control), 1 μg of RA0C_1193 mutagenic PCR fragments mixed with 1 μg of competing chromosomal DNA of E. coli XL1-Blue, or chromosomal DNA of R. anatipestifer ATCC 11845 as indicated for 1 h. The averages and standard deviations of three independent experiments are shown. The numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as the competing DNA and R. anatipestifer ATCC 11845 DNA as the competing DNA.

FIG 6

Confirmation of the presence of shuttle plasmid in R. anatipestifer ATCC 11845 transformants. Lane M, DNA ladder (Biomed, Beijing, China); lanes 1 to 9, XbaI-digested profile of extracted plasmids; lanes 1, 4, and 7, plasmids extracted from E. coli XL1-Blue; and lanes 2, 3, 5, 6, 8, and 9, plasmids extracted from R. anatipestifer ATCC 11845 transformants.