The bacterial strains and plasmids used in this study are listed in Table 1. R. anatipestifer strains were grown at 37°C in tryptic soybean broth (TSB, Oxoid) or tryptic soy agar (TSA, Oxoid) in an atmosphere of 5% CO₂. Escherichia coli (E. coli) strains were grown on Luria-Bertani (LB, Oxoid) broth or agar at 37°C. When required, antibiotics were added at the following final concentrations (μg/ml): Chloramphenicol (Cm, Sigma), 25; cefoxitin (Cfx, Sigma), 1; kanamycin (Kan, Sigma), 100; ampicillin (Amp, Sigma), 100 or spectinomycin (Spc, Sigma), 70. Diaminopimelic acid (DAP, 50 μg/ml) to E. coli X7213λpir cultures (Edwards et al., 1998).

For this study, 192 R. anatipestifer isolates were collected from different regions of China. All isolates were identified using the Biolog Microbial Identification System (Biolog, Hayward, CA, USA), as well as PCR and biochemical analyses (data not shown). After lysing the bacteria in lysis buffer (0.5% NP-40, Sigma; 200 ng/ml proteinase K, Takara Biotechnology Co., Ltd. Dalian, China), the presence of the cat gene was determined by PCR analysis using primers cat-F1 and cat-R1 (Table 2).

The cat genes were deleted by homologous recombination using a suicide vector pRE112 (Kong et al., 2011) as described previously (Luo et al., 2015). Briefly, the right flanking sequence (~620 bp) and the left flanking sequence (~620 bp) of the target genes G148_1769 and G148_1772 were amplified using primers 1769up-F and 1769up-R, 1769down-F and 1769down-R, 1772up-F and 1772up-R, and 1772down-F and 1772down-R, respectively (Table 2). The 1,145 bp SpcR cassette and the 1,192 bp CfxR cassette were amplified from plasmid pYES1 (Luo et al., 2015) and pLMF01 (Liu et al., 2016) using primers Spc-F, Spc-R and Cfx-F, Cfx-R, respectively (Table 2). The SpcR cassette and the CfxR cassette were used for deletion of G148_1769 and G148_1772, respectively. The PCR fragments were overlapped using the PCR method (Xiong et al., 2006). The fused PCR fragments were ligated to suicide plasmid pRE112, respectively, to produce pRE112:: 1769USD (SpcR) and pRE112:: 1772UCD (CfxR). Subsequently, the recombinant plasmids were introduced into R. anatipestifer CH-2 by conjugation as described previously (Liao et al., 2015). The transconjugants were selected on TSA plates supplemented with Spc (40 μg/ml) or Cfx (1 μg/ml). The gene-deletion mutant strains, which were designated as RA-CH2Δ1769, RA-CH2Δ1772, and RA-CH2Δ1769Δ1772, were identified by PCR analysis.

The cat^H79A mutant was constructed by in vitro site-directed mutagenesis. The upstream and downstream mutated regions of the cat gene of R. anatipestifer CH-2 amplified using primers MF1, MR1 and MF2, MR2 his, respectively (Table 2). The fragments were fused by overlap extension PCR to yield the mutant gene cat^H79A.

Complete cat and cat^H79A genes were amplified by PCR from R. anatipestifer CH-2 chromosomal DNA and by in vitro site-directed mutagenesis using primers catF2 and catR2, catF2, MR1 and MF2, catR2 (Table 2), for complementation. Complete cat and cat^H79A genes were amplified by PCR from R. anatipestifer CH-2 chromosomal DNA and by in vitro site-directed mutagenesis using primers MF1 and MR2 his, MF1, MR1 and MF2, MR2 his, respectively (Table 2), for expression of CAT and CAT^H79A proteins. The complementation fragments were purified and digested with NcoI and XhoI, and ligated to the pLMF02 plasmid digested with NcoI and XhoI. The expression fragments were purified and digested with NdeI and XhoI, and ligated with the pET30a plasmid digested with corresponding restriction endonucleases. The ligation mixtures were introduced into CaCl₂-competent DH5α cells. Transformants were screened by PCR, and positive clones were sequenced.

The plasmids, pLMF02, pLMF02:: cat, and pLMF02:: cat^H79A, were introduced into R. anatipestifer ATCC 11845, respectively, by the method described previously (Liao et al., 2015). The transconjugants were selected on TSA plates supplemented with Cfx (1 μg/ml) and Kan (40 μg/ml). The complementation strains, RA-ATCC11845 (pLMF02), RA-ATCC11845 (pLMF02:: cat), and RA-ATCC11845 (pLMF02:: cat^H79A), were identified by PCR analysis.

Strains E. coli BL21 (DE3) (pET30a:: cat-s) and E. coli BL21 (DE3) (pET30a:: cat^H79A -s) were grown overnight in LB medium containing Kan (100 μg/ml). Stationary-phase cultures were diluted to an OD₆₀₀ of 0.05 in 500 ml of LB medium containing Kan (100 μg/ml) and incubated with shaking at 37°C until the culture density reached an OD₆₀₀ of 0.6. Cells were then induced with 0.4 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and reincubated at 37°C. The cells were harvested by centrifugation for 10 min at 8,000 rpm at 4°C, and then the pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0; 50 ml) and sonicated. The cell lysate was clarified by centrifugation to eliminate cell debris and then applied to a metal affinity resin column that was equilibrated with the same buffer. The column was successively washed with buffers containing 20 mM, 50 mM imidazole, and phosphate buffer (pH 4.4 and 5.0, respectively). Recombinant proteins were ultrafiltered with storage buffer (20 mM Tris-HCl, pH 7.8). The protein purity was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining. Protein concentration was determined using the BCA method with bovine serum albumin as the standard.

Chloramphenicol MIC tests for deletion mutants and complementary strains were performed in 96-well microtiter plates according to the Clinical and Laboratory Standard Institute criteria (CLSI, 2015). E. coli ATCC 25922 was used as a quality-control strain. The turbidity of the inocula was adjusted to 10⁷ CFU/ml (100 μl/well). An inoculated broth containing no antibiotics was included as positive control, and a tube of uninoculated broth was used as negative control. The experiments were repeated three times.

To assess whether the cat gene of R. anatipestifer was regulated by chloramphenicol, the wild-type strain was grown with TSB with or without 1 μg/ml of chloramphenicol. Total RNA was isolated from strains grown to log phase (OD₆₀₀ ≈ 0.8–1.0) by using the RNAiso Plus kit (TaKaRa). DNA was removed using RNase-Free DNase. cDNA was generated by using the Sensiscript RT kit (TaKaRa), according to the manufacturer's instructions. Real-time quantitative PCR (qPCR) was performed to measure cat mRNA levels using SYBR Premix EX Taq II (TaKaRa). The primers used in real-time qPCR analysis are listed in Table 2. The expression level of the cat gene was normalized to that of the recA gene, which was used as reference. All PCR reactions were performed in triplicate. The efficiency of primer binding was determined by linear regression by plotting the cycle threshold (CT) value vs. the log of the cDNA dilution. Relative quantification of the transcript was determined using the comparative CT method (2^−ΔΔCT), calibrated to recA. The experiments were performed multiple times independently and generated comparable results. The findings are presented as fold-change relative to the mRNA expression levels of the control strains.

CAT catalyzes the transfer of an acetyl group from acetyl-CoA to Cm, producing acetylated Cm and CoASH. The CATase activity was assayed based on the disappearance of acetyl-CoA during Cm acetylation (Kobayashi et al., 2015). The reaction mixture contained 0.25 ml of 0.2 M Tris-HCl (pH 7.8), 0.05 ml of 1 mM acetyl-CoA, 0.05 ml of 1 mM Cm, 0.05 ml of 10 mM DTNB [5,5′-dithio-bis (2-nitrobenzoic acid)], and 0.1 ml enzyme extract. The reaction was initiated by the addition of Cm. An increase in absorbance at a wavelength of 412 nm, which arises from 5-thio-2-nitrobenzoic acid, was derived from the reaction between free CoASH and DTNB. The concentration of 5-thio-2-nitrobenzoic acid was determined using its molar extinction coefficient at 412 nm (13,600 M⁻¹ cm⁻¹). The value was then used in the determination of the amount of CoASH produced during the reaction. One unit of enzyme activity is defined as the amount of activity catalyzing 1 μmol of acetyl transfer per min under the assay conditions.

The changes of mRNAs were expressed as fold expression and calculated using the comparative CT (2^−ΔΔCT) method. The results of RT-PCR were performed using GraphPad Prism 6.0 software for Windows (GraphPad Software Inc., La Jolla, USA). Homology analysis of types A and B CATs based on amino acid identity using DNAMAN 8.0 (Lynnon-Biosoft, Ontario, Canada).

The cat gene was identified in 69/192 (36%) R. anatipestifer isolates collected from China, thereby suggesting that the cat gene was widely distributed among R. anatipestifer strains. Sequence analysis found that there are two copies of the cat gene (G148_1769: 1854900…1855529 and G148_1772: 1858427…1859056) in R. anatipestifer CH-2. There is no similarity between the cat gene from R. anatipestifer CH-2 and type A cat genes previously reported. However, the cat genes share 99–100% identity in R. anatipestifer strains reported in NCBI.

To verify whether the cat genes of R. anatipestifer CH-2 were responsible for chloramphenicol resistance, the deletion strains and complementation strains were constructed. Table 3 showed that the chloramphenicol MICs of R. anatipestifer CH-2 and RA-CH2Δ1769 were 32 and 64 μg/ml, respectively. Compared to the MIC of the wild-type strain, the MIC of RA-CH2Δ1769 increased (Table 3). Similarly, another signal cat gene deletion strain RA-CH2Δ1772 had no obviously decreased in resistance to chloramphenicol, exhibiting 32 μg/ml in the minimum inhibitory concentration for chloramphenicol (Table 3). Thus, we supposed that the two copies of cat gene in R. anatipestifer CH-2 were involved in chloramphenicol resistance. The two copies of the cat gene deletion strain RA-CH2Δ1769Δ1772 was constructed. The level of chloramphenicol resistance was determined to be significantly reduced, 4 μg/ml.

To further verify that the cat genes are related to chloramphenicol resistance in R. anatipestifer, shuttle plasmid pLMF02 with the cat gene was introduced into R. anatipestifer ATCC 11845, which is sensitive to chloramphenicol. It was restored the level of chloramphenicol resistance (Table 3). These results strongly suggested that the cat gene was responsible for chloramphenicol resistance in R. anatipestifer.

According to the study described above, the minimum inhibitory concentrations for chloramphenicol between RA-CH2Δ1769 and RA-CH2Δ1772 are not same (Table 3). To explore whether the transcription of cat gene is affected by single deletion strain, RT-PCR analysis was performed. The result revealed that G148-1772 was upregulated 3.82-fold in the RA-CH2Δ1769 mutant (Figure 1). However, the mRNA level of G148-1769 in RA-CH2Δ1772 did not increased significantly. This information could explain why the resistance level of RA-CH2Δ1769 is greater than RA-CH2Δ1772 and wild-type strain. The result showed that the cat genes do mediate the production of chloramphenicol resistance and the relationship of the two cat copies is complementary and cooperative in R. anatipestifer CH-2.

In order to study the cellular strategies used by R. anatipestifer CH-2 and mutant strains in the presence of chloramphenicol, we decided to analyze transcriptional changes of cat gene in these strains growing in the presence of 1 μg/ml of this antibiotic. Chloramphenicol treatment had no bactericidal effect when sub-inhibitory concentration of chloramphenicol were applied (data not shown). We found that the mRNA level of cat genes was increased 11-fold, 13.94- and 18.31-fold in wild strain and mutant strains RA-CH2Δ1769, RA-CH2Δ1772, respectively (Figure 2). These results suggested that cat genes were regulated by chloramphenicol.

In a previous study involving Pseudomonas aeruginosa, His79 served as a major catalytic residue (Beaman et al., 1998). The two amino acid sequences of cat from P. aeruginosa and R. anatipestifer, respectively, showed 86.95% identity (Figure 3). To examine the main catalytic site of CAT from R. anatipestifer CH-2, the cat gene and the cat^H79A gene were expressed in E. coli cells. The gene products, which contained C-terminal His₆-tag, were purified by Ni-agarose affinity chromatography, yielding a distinct protein band in the SDS-PAGE gel, with an approximately molecular weight of ~25 kDa. The catalytic activities of CAT and CAT^H79A were analyzed at 37°C. The detailed information of the reaction mixtures is described in the Materials and Methods. The specific activities of CAT and CAT^H79A were 8.33 ± 0.38 and 0 U·mg⁻¹, respectively (Table 4). Meanwhile, the MIC of ATCC 11845 harbored cat^H79A was significantly lower than that of ATCC 11845 carried the cat gene (Table 3). Thus, the H79A substitution had a significant effect on CAT activity.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.