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. anatipestifer.
IMPORTANCE Riemerella 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
INTRODUCTION
Riemerella anatipestifer is a Gram-negative, non-spore-forming, rod-shaped bacterium that is a major causative agent of septicemia in waterfowl, turkey, and other birds (1). R. anatipestifer infection leads to great economic losses due to its high mortality in ducklings and poor feed conversion (2). At present, at least 21 serotypes of R. anatipestifer have been identified (3). However, the molecular mechanisms of infection remain largely unknown.
At present, complete genome sequences are available for several strains, including R. anatipestifer ATCC 11845, RA-GD, RA-CH-1, RA-CH-2, and Yb2 (4–6). Comparative genomics of RA-CH-1, RA-CH-2, and ATCC 11845 revealed that RA-CH-1 was 140,000 bp larger than the two other strains (4), suggesting that foreign DNA was acquired by this bacterium (4). DNA transfers from one bacterium to another via the conjugation process (7), bacteriophages (8), or natural transformation (9). Bacteria, such as Haemophilus influenzae and Neisseria meningitidis, can acquire foreign DNA through the natural transformation process (10, 11). Natural transformation is a major horizontal gene transfer (HGT) mechanism that is important for bacterial genetic diversification (12). Naturally competent bacteria take up exogenous DNA and integrate it into their chromosomes.
In R. anatipestifer, targeted mutants have been constructed by introducing recombinant DNA into the cells by conjugation (13). However, this method requires cloning steps and is tedious and poorly efficient. In this report, we investigated the natural transformation of R. anatipestifer and used this property to design an easy method to knock out genes in this bacterium.
RESULTS
Natural competence in R. anatipestifer ATCC 11845.
To investigate whether R. anatipestifer ATCC 11845 was naturally competent, we first cultured R. anatipestifer ATCC 11845 in GC broth (GCB) medium to exponential phase. A 300-μl sample of the culture (the optical density at 600 nm [OD600] was adjusted to 1) was mixed with 1 μg of RA-CH-1 erythromycin resistance (Ermr) chromosomal DNA (13, 14). After 1 h of incubation, 100 μl of the bacteria was spread on the Ermr plate. At the same time, the bacteria were also spread on the plates without an antibiotic to count the number of viable bacteria. This procedure gave abundant resistant colonies with a transformation frequency of 3.5 × 10−8 Ermr colonies. No resistant colonies appeared in the control experiment performed without the addition of DNA (the spontaneous mutation rate for erythromycin resistance is 10−10 colonies for R. anatipestifer ATCC 11845). Then, 6 Ermr colonies were isolated on the new GCB agar plate with erythromycin, and the genomes of the grown clones were extracted and used to perform PCR and sequencing. The results showed that the erythromycin resistance gene was inserted into the R. anatipestifer ATCC 11845 genome (Fig. 1). This result indicated that R. anatipestifer ATCC 11845 had natural competence in GCB medium.
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).
To investigate whether natural transformation occurs in any growth phase and natural transformation frequency in the different growth phases, the experiments were performed in the lag phase, exponential phase, stationary phase, and death phase as shown in Fig. S1 in the supplemental material. To avoid biases related to the variability of cell density, the growing bacteria were adjusted to an OD600 of 1 and were subsequently treated for transformation. Standardization of cell density was achieved by concentration or dilution with the culture supernatant. One microgram of RA-CH-1 Ermr chromosomal DNA was mixed with 300 μl (OD600 = 1) of the different growth phase bacteria. After incubation for 1 h at 37°C, the bacteria were spread on the GCB plate with or without antibiotic. We observed R. anatipestifer ATCC 11845 to be competent in all of the growth phases, although the transformation frequency was about 20 times lower for bacterial cells in the death phase compared to the transformation frequency of cells in the exponential growth phase (Table 1).
TABLE 1.
Natural transformation in different growth phases
| Growth phase | OD600 | Transformation frequency |
|---|---|---|
| Lag phase | 0.2 ± 0.05 | 4.2 (±0.8) × 10−9 |
| Exponential phase | 1.4 ± 0.3 | 6.7 (±1.6) × 10−8 |
| Stationary phase | 3.3 ± 0.2 | 9.1 (±2.3) × 10−9 |
| Death phase | 4.5 ± 0.7 | 3.9 (±1.2) × 10−9 |
Establishment of an easy knockout method in R. anatipestifer ATCC 11845.
Based on initial observations that natural transformation can be used to transfer chromosomal DNA between R. anatipestifer cells, we asked whether this method can be used for targeted mutagenesis using PCR fragments. We tested seven gene disruption constructs (RA0C_1193::Ermr, RA0C_1551::Ermr, RA0C_1534::Ermr, RA0C_1180::Ermr, RA0C_1563::Ermr, RA0C_1564::Ermr, and RA0C_0783::Ermr). All of the constructs contained the erythromycin resistance cassette from RA-CH-1 flanked by approximately 800 bp of R. anatipestifer ATCC 11845 sequences. The erythromycin resistance cassette of RA-CH-1 has been identified in our previous study (14), and it has been used to knock out tonB of R. anatipestifer ATCC 11845 (13). Here, only RA0C_1193::Ermr was taken as an example. After transformation with the PCR fragments, the cells were selected based on antibiotic resistance. The construct gave Ermr transformants at very high frequencies (∼5 × 104 transformants in ∼5 × 109 bacteria, with transformation frequencies of 10−5 transformants). Colonies were screened by analytical PCR to verify that they carried the appropriate insertions. The PCR tests validated the insertion of the element at the appropriate genomic site and the loss of sequences corresponding to the wild-type sequence (Fig. 2). Therefore, these data showed that natural transformation can be used to efficiently generate targeted gene disruptions in R. anatipestifer ATCC 11845. Additionally, we attempted to knock out the R. anatipestifer RA-CH-1 B739_0933 gene using natural transformation with a spectinomycin (Spc)-resistant gene from the plasmid pAM238 as a selection marker (15) since R. anatipestifer RA-CH-1 had erythromycin resistance (14, 16). This natural transformation was also successful, with a transformation frequency of 10−8 transformants. The analytical PCR was used to verify that they carried the appropriate insertions (see Fig. S2 in the supplemental material).
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 Ermr cassette was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers Ermr P1 and Ermr P2 (lane 3); the left flanking sequence of RA0C_1193 and the Ermr cassette was amplified from wild-type strain R. anatipestifer ATCC and mutant strain R. anatipestifer ATCCΔRA0C_1193 using primers RA0C_1193up P1 and Ermr P2 (lane 4); the Ermr 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 Ermr P1 and RA0C_1193down P2 (lane 5).
To evaluate the effect of transformant DNA length, we designed donor DNA in which homologous DNA regions flanking a marker gene varied in size. The fragments were amplified from R. anatipestifer ATCC 11845 ΔRA0C_1193, in which the erythromycin resistance gene had been stably inserted. As Table 2 showed, that transformation frequency was decreased when the flanking fragments were shorter. The transformation frequency was increased from 10−8 to 10−5 when the flanking fragment was increased from 50 bp to 800 bp (Table 2). When the bacteria were transformed with the PCR products providing 50-bp-long homologous DNA regions, transformation still occurred. The erythromycin resistance gene in the genome of the transformants was checked by PCR as described above (data not shown). It indicated that transformation efficiencies were directly related to the size of the homologous DNA region.
TABLE 2.
Effect of the length of flanking homologous DNA regions on transformation efficiency
| Length of the RA0C_1193 flanking region (bp) | Transformation frequency |
|---|---|
| 50 | 1.0 (±0.2) × 10−9 |
| 100 | 5.0 (±1.1) × 10−8 |
| 300 | 1.9 (±0.8) × 10−7 |
| 500 | 1.1 (±0.4) × 10−6 |
| 800 | 2.0 (±1.5) × 10−5 |
Natural transformation efficiency is dependent on the donor DNA concentration.
To study the effect of the DNA concentration on the transformation efficiency, we transformed R. anatipestifer ATCC 11845 with various amounts of RA0C_1193 mutagenic PCR fragments. Using competent cell preparations from the exponential phase containing 1.5 × 1010 CFU/ml, the DNA fragments (0.0001, 0.001, 0.01, 0.1, 0.2, 0.5, 1, 2, and 4 μg) were, respectively, added as indicated in Fig. 3. For DNA fragments between 0.1 ng and 1 μg, the transformation efficiency increased as the donor DNA concentration increased (Fig. 3). Figure 3 showed that the highest transformation efficiency was obtained at saturating DNA fragment concentrations (1 μg). The transformation efficiency obtained in the sample at a concentration of 0.0001 μg of DNA per ml (1 ± 0.1 × 105 transformants per μg of DNA) was greater than the efficiency obtained at a concentration of 1.0 μg of DNA per ml (4.5 ± 0.08 × 104 transformants per μg of DNA).
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. Ermr 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.
Natural transformation occurs within 1 min in R. anatipestifer ATCC 11845.
To examine the time required for the natural transformation process in R. anatipestifer ATCC 11845, aliquots of a R. anatipestifer ATCC 11845 culture grown to exponential phase were distributed into 9 samples, and each sample (300 μl; OD600 = 1) was equally transformed with 1 μg of the RA0C_1193 mutagenic PCR fragments as described in the Materials and Methods. Individual transformations were stopped at various time points (0 to 30 min) by the addition of DNase I and incubation for 1 h at 37°C. The transformants were scored by plating on selective agar. As a control, no transformants were obtained when DNase I was immediately added in the mixture. The addition of DNase I did not have any effect on the survival of R. anatipestifer ATCC 11845. Figure 4 showed that Ermr transformants were obtained after 1 min of exposure to DNA, and a saturated level of transformants was obtained after approximately 20 min of cocultivation with DNA (Fig. 4). These results showed that the natural transformation process of R. anatipestifer ATCC 11845 was able to proceed to completion within 1 min. This process was quicker than that of Ralstonia solanacearum (17).
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. Ermr transformants were selected after 1 h of incubation.
R. anatipestifer ATCC 11845 preferentially takes up its own DNA compared to Escherichia coli DNA.
Naturally competent species, such as Pasteurellaceae, take up their own DNA in preference to heterogeneous DNA (18). We examined whether R. anatipestifer ATCC 11845 also preferentially took up its own DNA using transformation competition experiments. Exponential-phase competent R. anatipestifer ATCC 11845 cells were incubated with 1 μg of E. coli XL1-Blue genomic DNA and 1 μg of RA0C_1193 mutagenic PCR fragments or 1 μg of R. anatipestifer ATCC 11845 genomic DNA and 1 μg of RA0C_1193 mutagenic PCR fragments as described in the Materials and Methods. After incubation for 1 h at 37°C, the bacteria were spread on the selective plates (GCB agar plates with 1 μg/ml erythromycin), and the transformation efficiency was calculated. As expected, we observed a significant reduction in the transformation efficiency for the sample to which R. anatipestifer ATCC 11845 genomic DNA was added but not the sample to which E. coli genomic DNA was added (Fig. 5). To exclude the possibility that the genomic DNA was degraded by R. anatipestifer ATCC 11845, we took a sample at different time points after genomic DNA was mixed with R. anatipestifer ATCC 11845 and performed electrophoresis. The result showed that there was no significant degradation of either E. coli or R. anatipestifer ATCC 11845 genomic DNA (Fig. S2). It was confirmed that R. anatipestifer ATCC 11845 DNA competed with the RA0C_1193 mutagenic PCR fragments for uptake. In contrast, the addition of unmarked E. coli DNA did not significantly alter transformation efficiency (Fig. 5). Therefore, E. coli DNA competes poorly with R. anatipestifer ATCC 11845 DNA for uptake, and R. anatipestifer ATCC 11845 preferentially takes up its own DNA compared to E. coli DNA (Fig. 5). These data suggested that the natural transformation of R. anatipestifer is species specific.
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.
Transformation with plasmid DNA.
Transformation with circular plasmid DNA was tested with shuttle plasmid pLMF03 (19) and shuttle plasmids containing R. anatipestifer ATCC 11845 fragments, pLMF03::tonB1 and pLMF03::tonB2 (19). These shuttle plasmids contain the cefoxitin resistance gene (Cfxr). Exponential-phase competent R. anatipestifer ATCC 11845 cells (300 μl; OD600 = 1) and 1-μg plasmids were used to perform the experiments. The bacteria were spread on the selective plates, and the transformation efficiency was calculated. The result showed that Cfxr transformants were obtained with a transformation efficiency of 20 to 30 transformants for 1 μg of empty pLMF03 plasmid. However, Cfxr transformants were obtained with a transformation efficiency of ∼103 transformants for 1 μg of pLMF03::tonB1 or 1 μg of pLMF03::tonB2. To exclude the Cfxr clones that were from spontaneous mutation, the plasmids were extracted from these colonies. The presence of pLMF03, pLMF03::tonB1, or pLMF03::tonB2 plasmid DNA was verified by restriction digestion by XbaI and electrophoreses (Fig. 6).
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.
Searching for the components of the natural transformation machinery in R. anatipestifer genomes.
To investigate whether R. anatipestifer contained the genes required for DNA uptake, we searched the genome sequence of R. anatipestifer ATCC 11845 for homologues of the natural transformation machinery encoded by H. influenzae (20) and Neisseria (21). We found no homologues of the gene that encoded the typical pilus structure, but we did identify some homologues for ComE, ComM, DprA, RadC, and Ssb (Table 3).
TABLE 3.
Homologues of the H. influenzae and N. gonorrhoeae natural transformation machinery in R. anatipestifer ATCC 11845
| H. influenzae category and homolog |
R. anatipestifer ATCC 11845 |
N. gonorrhoeae locus identity |
R. anatipestifer ATCC 11845 |
||
|---|---|---|---|---|---|
| Locus identity | Identity (%)a | Locus identity | Identity (%)b | ||
| DNA uptake | |||||
| ComB (HI_0438) | Absent | ComE (NGO_1178) | RA0C_0821 | 39 | |
| DNA processing | |||||
| ComM (HI_1117) | RA0C_1532 | 47 | ComM (NGO_1550) | RA0C_1532 | 42 |
| DprA (HI_0985) | RA0C_1073 | 36 | DprA (NGO_1865) | RA0C_1073 | 31 |
| RadC (HI_0952) | RA0C_0744 | 36 | RadA (NGO_0367) | RA0C_0744 | 46 |
| Ssb (HI_0250) | RA0C_0532 | 43 | Ssb (NGO_1031) | RA0C_0532 | 39 |
This percentage indicates the sequence identity between R. anatipestifer ATCC 11845 and H. influenzae Rd KW20 predicted proteins.
This percentage indicates the sequence identity between R. anatipestifer ATCC 11845 and N. gonorrhoeae FA 1090 predicted proteins.
DISCUSSION
R. anatipestifer is one of the most important duck pathogens. Although at least 5 R. anatipestifer species have been sequenced, their pathogenic mechanism and evolution remain largely unknown. To investigate the existence of genetic exchanges between R. anatipestifer species, we performed natural transformation experiments using the similar protocol for Neisseria (22). As described in other bacteria, we found that natural transformation occurred between R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1. In other bacteria, such as H. influenzae, the cell is not constitutively competent. Instead, the cells become competent when nutrients are limited (23). In laboratory conditions, the cell competency of H. influenzae is induced when the bacteria are transferred from rich medium to starvation medium (24). In contrast, the natural transformation of R. anatipestifer occurs at all of the growth phases and did not need induction, although the transformation frequency was different in different growth phases.
Gene knockout in R. anatipestifer was previously produced by conjugation in our group (13) and other groups (25), which involved the transfer of a suicide vector from a donor bacterium (E. coli) to the recipient bacterium (R. anatipestifer). This method, which required cloning steps, was time-consuming and did not always work well (our unpublished data). Here, we exploited the natural competence of R. anatipestifer ATCC 11845 to develop a gene knockout method. The method was more simple and rapid than the conjugation method. The recombinant frequency was 10−6 or less resistant transconjugants using traditional conjugation (26; our unpublished data). The transformation frequency increased by at least 10-fold using natural transformation. At present, at least 7 gene replacement mutants have been generated in the R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1 strains by our group. The PCR fragments used were about 0.8 kb in length and appear to be optimal. Additionally, the gene knockout was still successful when using the homologous regions of ∼50 bp. Thus, this sequence can be directly incorporated in the PCR primer, which would greatly facilitate mutagenesis.
It has been demonstrated that H. influenzae and N. gonorrhoeae prefer to take up the DNA contained in a specific sequence, the DNA uptake sequence (DUS) (21). Here, R. anatipestifer ATCC 11845 DNA competes efficiently with E. coli DNA for uptake by R. anatipestifer ATCC 11845. Moreover, the shuttle plasmid containing its own fragment more easily entered the cells than the empty plasmid, suggesting that the fragment may contain a specific sequence for uptake. The specific sequence for uptake of R. anatipestifer should be identified in future study.
In summary, our natural transformation studies in R. anatipestifer provided a new avenue for genetic engineering in this bacterium. We have established a procedure for the efficient construction of targeted mutants that will facilitate the identification and characterization of R. anatipestifer virulence factors and eventually contribute to a better understanding of R. anatipestifer pathogenicity.
MATERIALS AND METHODS
Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are shown in Table S1 in the supplemental material.
Media and growth conditions.
R. anatipestifer was routinely cultured in liquid cultures in GC broth (GCB) (1.5% proteose peptone [Oxoid, China], 0.4% K2HPO4 [Sigma], 0.1% KH2PO4 [Sigma], and 0.5% NaCl [Sigma] plus Kellogg's supplements I, 40% glucose [Sigma], 1% l-glutamine [Sigma], 0.002% thiamine pyrophosphate [Sigma], and Kellogg's supplements II, 0.5% Fe(NO3)3 [Sigma]) (27, 28) at 37°C with agitation. GCB agar plates were prepared by GCB supplementation with 1.5% agar at 37°C. Antibiotics were added to the following final concentrations (μg/ml): spectinomycin (Spc), 80; erythromycin (Erm), 1; and cefoxitin (Cfx), 1 for R. anatipestifer. Ampicillin (Amp) was added to a final concentration of 100 μg/ml for E. coli. The bacteria were grown in broth with shaking at 180 rpm at 37°C.
In vitro growth rate determination.
The in vitro growth rates of the tested strains were determined by measuring the OD600 with a spectrophotometer (BioPhotometer; Eppendorf, Germany). Briefly, cultures in the early exponential phase were inoculated into 20 ml of GCB medium at an OD600 of 0.05 and incubated at 37°C with shaking at 180 rpm. The OD600 was determined every 2 h for 14 h. At the same time points, viable bacteria were quantified by plating serial dilutions onto GCB agar plates.
Natural transformation.
Natural transformation was performed as described previously for N. meningitidis (22) with a little modification. Briefly, the bacterial cultures grown in liquid GCB were collected and the OD600 was checked. Then, the bacteria were centrifuged at 8,000 × g for 10 min and the supernatant was removed. The pellet was suspended in fresh GCB buffer to adjust the OD600 to 1. The bacterial suspensions (0.3 ml) were transferred to sterilized tubes, and 1 μg of RA-CH-1 genomic DNA or DNA fragments carrying the erythromycin resistance gene (Ermr) (13) were added to the tube. After additional incubation for 1 h at 37°C, the bacterial cultures were serially diluted and plated onto GCB agar plates with or without antibiotics.
Transformation frequency and transformation efficiency assays.
The recipient bacteria were grown in GCB liquid medium. The DNA suspension was added to 300 μl (OD600 = 1) of bacterial suspension. The mixture was incubated for an additional 1 h at 37°C. The bacterial suspensions were diluted and plated onto a selective medium for 1 day at 37°C. A suspension without DNA was used as the negative control. The transformation frequency was calculated as the number of transformants per CFU (29), and the transformation efficiency was calculated as the number of transformants per microgram of DNA (30).
Competition assays.
Transforming DNA (1 μg for each sample) and competing DNA (1 μg of genomic DNA of R. anatipestifer ATCC 11845 or 1 μg of genomic DNA of E. coli strain XL1-Blue) were mixed and added to the competent bacteria (300 μl; OD600 = 1). After 1 h of incubation at 37°C, the mixtures were spread on the plates with or without antibiotics. The transformation efficiency was calculated.
DNA manipulation.
Small-scale plasmid DNA preparations were generated using a Tianprep mini plasmid kit (Tiangen, Beijing, China). Bacterial chromosomal DNA was extracted using the Tianamp bacteria DNA kit (Tiangen, Beijing, China). XbaI restriction enzyme cutting was performed according to the manufacturer's recommendations (NEB, China). The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA). Purification of DNA fragments from the PCR and the restriction reaction was performed using the Tiangen extract II kit (Tiangen, Beijing, China).
Generation of mutagenic PCR products.
Mutagenic PCR fragments were created by joining three fragments corresponding to the regions flanking the sequence to be deleted and a gene encoding antibiotic resistance. Here, we use the RA0C_1193 gene as an example to describe the process. Briefly, an approximately 800-bp left flanking sequence and an 800-bp right flanking sequence of the R. anatipestifer ATCC 11845 RA0C_1193 gene were amplified by PCR using the primer pairs RA0C_1193 upP1/RA0C_1193 upP2 and RA0C_1193 downP1/RA0C_1193 downP2, respectively (see Table S2 in the supplemental material). A 994-bp sequence containing the Ermr cassette was amplified from the genome of R. anatipestifer RA-CH-1 (13) using the primers Ermr P1 and Ermr P2 (Table S2). Three PCR fragments (RA0C_1193 upstream, RA0C_1193 downstream, and the Ermr cassette) were amplified using the overlap PCR method (31). The same process was used for the construction of other mutagenic PCR products. For the RA-CH-1 B739_0933 mutant, a 1,140-bp sequence containing the SpcR cassette from shuttle plasmid pAM238 (15) was used as a selected marker. The respective primers are shown in Table S2.
Natural transformation-based knockout.
The R. anatipestifer ATCC 11845 strains were grown in GCB medium at 37°C. The bacterial density was adjusted to an OD600 of 1. Three hundred microliters of the bacterial suspension was placed inside a 1.5-ml sterilized tube, supplemented with a PCR fragment (1 μg), and incubated for 1 h at 37°C. Then, 100-μl samples of the mixture were plated onto plates supplemented with selective antibiotics and incubated overnight at 37°C. Six clones were isolated on selective medium and screened using PCR.
Statistical analysis.
The statistical analysis was performed using GraphPad Prism 6 software for Windows. The statistical significance of the data was ascertained using Student's t test. A P value of <0.05 was considered significant.
Accession number(s).
The nucleotide sequences of R. anatipestifer ATCC 11845, RA-CH-1, and RA-CH-2 were deposited in GenBank under accession numbers CP003388, CP003787, and CP004020, respectively.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the International S&T Cooperation Program of Sichuan Province (grant 2016HH0052), the National Natural Science Foundation of China (grant 31302131; http://www.nsfc.gov.cn/), the National Science and Technology Support Program (2015BAD12B05), the China Agricultural Research System (CARS-43-8), and the Integration and Demonstration of Key Technologies for Duck Industrialization in Sichuan Province (2014NZ0030).
M.L. and A.C. conceived and designed the experiments. L.Z., M.L., L.H., and D.Z. performed the experiments. M.W., R.J., S.C., and K.S. analyzed the data. Y.W., Q.Y., and X.C. contributed reagents/materials/analysis tools. M.L., F.B., and A.C. wrote the paper. All authors have reviewed the manuscript.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00127-17.
REFERENCES
- 1.Leavitt S, Ayroud M. 1997. Riemerella anatipestifer infection of domestic ducklings. Can Vet J 38:113. [PMC free article] [PubMed] [Google Scholar]
- 2.Turbahn A, De Jackel SC, Greuel E, De Jong A, Froyman R, Kaleta EF. 1997. Dose response study of enrofloxacin against Riemerella anatipestifer septicaemia in Muscovy and Pekin ducklings. Avian Pathol 26:791–802. doi: 10.1080/03079459708419253. [DOI] [PubMed] [Google Scholar]
- 3.Pathanasophon P, Phuektes P, Tanticharoenyos T, Narongsak W, Sawada T. 2002. A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailand. Avian Pathol 31:267–270. doi: 10.1080/03079450220136576. [DOI] [PubMed] [Google Scholar]
- 4.Wang X, Liu W, Zhu D, Yang L, Liu M, Yin S, Wang M, Jia R, Chen S, Sun K, Cheng A, Chen X. 2014. Comparative genomics of Riemerella anatipestifer reveals genetic diversity. BMC Genomics 15:479. doi: 10.1186/1471-2164-15-479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Wang X, Zhu D, Wang M, Cheng A, Jia R, Zhou Y, Chen Z, Luo Q, Liu F, Wang Y, Chen XY. 2012. Complete genome sequence of Riemerella anatipestifer reference strain. J Bacteriol 194:3270–3271. doi: 10.1128/JB.00366-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wang X, Ding C, Wang S, Han X, Yu S. 2015. Whole-genome sequence analysis and genome-wide virulence gene identification of Riemerella anatipestifer strain Yb2. Appl Environ Microbiol 81:5093–5102. doi: 10.1128/AEM.00828-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Willetts N, Skurray R. 1980. The conjugation system of F-like plasmids. Annu Rev Genet 14:41–76. doi: 10.1146/annurev.ge.14.120180.000353. [DOI] [PubMed] [Google Scholar]
- 8.Menouni R, Hutinet G, Petit MA, Ansaldi M. 2015. Bacterial genome remodeling through bacteriophage recombination. FEMS Microbiol Lett 362:1–10. [DOI] [PubMed] [Google Scholar]
- 9.Stewart GJ, Carlson CA. 1986. The biology of natural transformation. Annu Rev Microbiol 40:211–235. doi: 10.1146/annurev.mi.40.100186.001235. [DOI] [PubMed] [Google Scholar]
- 10.Aas FE, Wolfgang M, Frye S, Dunham S, Lovold C, Koomey M. 2002. Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol Microbiol 46:749–760. doi: 10.1046/j.1365-2958.2002.03193.x. [DOI] [PubMed] [Google Scholar]
- 11.MacFadyen LP, Chen D, Vo HC, Liao D, Sinotte R, Redfield RJ. 2001. Competence development by Haemophilus influenzae is regulated by the availability of nucleic acid precursors. Mol Microbiol 40:700–707. doi: 10.1046/j.1365-2958.2001.02419.x. [DOI] [PubMed] [Google Scholar]
- 12.Matthey N, Blokesch M. 2016. The DNA-uptake process of naturally competent Vibrio cholerae. Trends Microbiol 24:98–110. doi: 10.1016/j.tim.2015.10.008. [DOI] [PubMed] [Google Scholar]
- 13.Liao H, Cheng X, Zhu D, Wang M, Jia R, Chen S, Chen X, Biville F, Liu M, Cheng A. 2015. TonB energy transduction systems of Riemerella anatipestifer are required for iron and hemin utilization. PLoS One 10:e0127506. doi: 10.1371/journal.pone.0127506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Luo H, Liu M, Wang L, Zhou W, Wang M, Cheng A, Jia R, Chen S, Sun K, Yang Q, Chen X, Zhu D. 2015. Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer. Avian Pathol 44:162–168. doi: 10.1080/03079457.2015.1019828. [DOI] [PubMed] [Google Scholar]
- 15.Fournier C, Smith A, Delepelaire P. 2011. Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity. Mol Microbiol 80:133–148. doi: 10.1111/j.1365-2958.2011.07562.x. [DOI] [PubMed] [Google Scholar]
- 16.Xing L, Yu H, Qi J, Jiang P, Sun B, Cui J, Ou C, Chang W, Hu Q. 2015. ErmF and ereD are responsible for erythromycin resistance in Riemerella anatipestifer. PLoS One 10:e0131078. doi: 10.1371/journal.pone.0131078. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Bertolla F, Van Gijsegem F, Nesme X, Simonet P. 1997. Conditions for natural transformation of Ralstonia solanacearum. Appl Environ Microbiol 63:4965–4968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Redfield RJ, Findlay WA, Bosse J, Kroll JS, Cameron AD, Nash JH. 2006. Evolution of competence and DNA uptake specificity in the Pasteurellaceae. BMC Evol Biol 6:82. doi: 10.1186/1471-2148-6-82. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Liu M, Wang M, Zhu D, Wang M, Jia R, Chen S, Sun K, Yang Q, Wu Y, Chen X, Biville F, Cheng A. 2016. Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown. Sci Rep 6:37159. doi: 10.1038/srep37159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Dougherty BA, Smith HO. 1999. Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis. Microbiology 145:401–409. [DOI] [PubMed] [Google Scholar]
- 21.Hamilton HL, Dillard JP. 2006. Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination. Mol Microbiol 59:376–385. doi: 10.1111/j.1365-2958.2005.04964.x. [DOI] [PubMed] [Google Scholar]
- 22.Biville F, Brezillon C, Giorgini D, Taha MK. 2014. Pyrophosphate-mediated iron acquisition from transferrin in Neisseria meningitidis does not require TonB activity. PLoS One 9:e107612. doi: 10.1371/journal.pone.0107612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Spencer HT, Herriott RM. 1965. Development of competence of Haemophilus influenzae. J Bacteriol 90:911–920. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Macfadyen LP, Dorocicz IR, Reizer J, Saier MH Jr, Redfield RJ. 1996. Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system. Mol Microbiol 21:941–952. doi: 10.1046/j.1365-2958.1996.441420.x. [DOI] [PubMed] [Google Scholar]
- 25.Wang X, Yue J, Ding C, Wang S, Liu B, Tian M, Yu S. 2016. Deletion of AS87_03730 gene changed the bacterial virulence and gene expression of Riemerella anatipestifer. Sci Rep 6:22438. doi: 10.1038/srep22438. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Hu Q, Zhu Y, Tu J, Yin Y, Wang X, Han X, Ding C, Zhang B, Yu S. 2012. Identification of the genes involved in Riemerella anatipestifer biofilm formation by random transposon mutagenesis. PLoS One 7:e39805. doi: 10.1371/journal.pone.0039805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Yi K, Rasmussen AW, Gudlavalleti SK, Stephens DS, Stojiljkovic I. 2004. Biofilm formation by Neisseria meningitidis. Infect Immun 72:6132–6138. doi: 10.1128/IAI.72.10.6132-6138.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Kellogg DS Jr, Peacock WL Jr, Deacon WE, Brown L, Pirkle DI. 1963. Neisseria Gonorrhoeae. I. Virulence genetically linked to clonal variation. J Bacteriol 85:1274–1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Kristensen BM, Sinha S, Boyce JD, Bojesen AM, Mell JC, Redfield RJ. 2012. Natural transformation of Gallibacterium anatis. Appl Environ Microbiol 78:4914–4922. doi: 10.1128/AEM.00412-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Wang Y, Goodman SD, Redfield RJ, Chen C. 2002. Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans. J Bacteriol 184:3442–3449. doi: 10.1128/JB.184.13.3442-3449.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Xiong AS, Yao QH, Peng RH, Duan H, Li X, Fan HQ, Cheng ZM, Li Y. 2006. PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1:791–797. doi: 10.1038/nprot.2006.103. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.