Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer

Figures & data

Table 1. Strains, plasmids and primers used in this study.

Strain or plasmid	Description	Source or reference
Strain
R. anatipestifer CH-1	Serotype 1, erm^R, Spec^S
R. anatipestifer CH-1ΔermF	ermF deletion mutant of CH-1 strain, Spec^R	This study
E. coli K-12 X7232	endA1 hsdR17 (rK^-mk^+) glnV44 thi-1 recA1 gyrA relA1Δ(lacZYA-argF)U169λpir deoR (Φ80dlac Δ(lacZ)M15)	(Roland et al., 1999)
E. coli K-12 X7213	thi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1 RP4-2-Tc::Mu λpirΔasdA4Δzhf-2::Tn10	(Roland et al., 1999)
XL1-Blue	endA1 gyrA96 (nalR) thi-1 recA1 relA1 lac glnV44F'[ ::Tn10proAB ^+lacIq Δ (lacZ) M15] hsdR17(rK^- mK^+), host for plasmid expression vectors that utilize the pBAD promoter	Stratagene Corporation, La Jolla, CA, USA
Plasmid
pRE112	sacB mobRP4 R6K ori Cm^+, pRE112-T-vector	(Kong et al., 2011)
pYES1new	YAC-BAC shuttle plasmid with spectinomycin resistance	Invitrogen
pBAD24	E. coli expression vector containing the arabinose pBAD promoter	(Guzman et al., 1995)
Primers for construction of suicide plasmid
Erm-Left F	5′ CATTAGTTAAGTCAACAATATCGTATGGAGTAGAACCTGG 3′	This study
Erm-Left R	5′ TGTCCTGGCTGGTAGTAACTTCTTACAGGTGAATACT 3′	This study
Spc F	5′ TGTAAGAAGTTACTACCAGCCAGGACAGAAATGCC 3′	This study
Spc R	5′ CCCGACTTTGAATTATTTGCCGACTACCTTGGTG 3′	This study
Erm-Right F	5′ GTAGTCGGCAAATAATTCAAAGTCGGGTGGTTGTC 3′	This study
Erm-Right R	5′ AAAACCGTCTGAAAAAGAGTTTCAAAATCTAATAAACGAT 3′	This study
Primers for identification of ermF deletion
16s rRNA F	5′ CTTCGGATACTTGAGAGCG 3′	This study
16s rRNA R	5′ GCAGCACCTTGAAAATTGT 3′	This study
Erm F	5′ AGAAATTGCCCGTTCGTTTTACGG 3′	This study
Erm R	5′ ATTTTTCAGGGACAACTTCCAGCATTT 3′	This study
IdentEF	5′ GCTATTTGAGAAATTAGTCCGCCCAA 3′	This study
IdentER	5′ CGACTGCCCTGCTGCGTAACA 3′	This study
Primers for cloning of the ermF gene
Erm-N-EcoRI	5′ CCGGAATTCCGGATGACAAAAAAGAAATTGCCCG 3′ (EcoRI site underlined)	This study
Erm-C-KpnI	5′ CGGGGTACCCCGCTACGAAGGATGAAATTTTTCAGGGAC 3′ (KpnI site underlined)	This study

Figure 1. Identification of R. anatipestifer CH-1ΔermF mutant by PCR amplification. Samples were electrophoresed on a 1% agarose (w/v) gel. Wild-type R. anatipestifer CH-1 (lanes 1 and 7), CH-1ΔermF (lanes 2, 5, 8 and 11), plasmid pYES1 new (lane 4), plasmid pYA4278-RLS (lane 10) and distilled water (lanes 3, 6, 9 and 12) were used as template in PCR amplifications using the following sets of primers. Lanes 1–3: 16S rRNA F and 16S rRNA R, which amplify a 906-bp fragment from R. anatipestifer 16S rRNA. Lanes 4–6: Spc F and Spc R, which amplify a 1172-bp fragment from the Spec^R cassette. Lanes 7–9: Erm F and Erm R, which amplify a 777-bp fragment of ermF, indicating the ermF gene was entire deleted. Lanes 10–12: IdentEF and IdentER, which amplify a 1036-bp fragment from the Spec^R cassette, indicating it was inserted in the correct position in R. anatipestifer CH-1 genome. M: DNA Marker 2000.

Figure 2. Identification of the ermF gene in a subset of clinical R. anatipestifer isolates by PCR amplification. Samples were electrophoresed on a 1% agarose (w/v) gel. The ermF gene was amplified by PCR using primers Erm F and Erm R. Lane 1: R. anatipestifer CH-1 genomic DNA was used as positive control template. Lane 2–24: subset of clinical R. anatipestifer isolates. Lane 25: distilled water as negative control template. M: DNA Marker 2000.

Figure 3. Growth curves for R. anatipestifer CH-1 and CH-1ΔermF. R. anatipestifer CH-1 and CH-1ΔermF were cultured (1-ml seed inoculation, overnight) in 100-ml TSB, and the growth curves were determined.

Table 2. Antimicrobial susceptibility of R. anatipestifer CH-1ΔermF and parent strain.

	Minimum inhibitory concentration (µg/ml)
Strains	ERY	AZI	TYL	LIN	TET	FLO
RA CH-1	256	512	32	>1028	4	1
CH-1ΔermF	0.25	0.5	8	0.25	8	1

ERY, erythromycin; AZI, azithromycin; TYL, tylosin; LIN, lincomycin; TET, tetracycline; FLO, florfenicol.

Figure 4. Growth curves for E. coli XL1-blue (pBAD24) and E. coli XL1-blue (pBAD24-ermF). E. coli XL1-blue (pBAD24) and E. coli XL1-blue (pBAD24-ermF) were grown in LB with or without erythromycin (ERY) and with or without 0.4% arabinose (ara).

Kong, Q., Yang, J., Liu, Q., Alamuri, P., Roland, K.L. & Curtiss, R. (2011). Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar Typhimurium. Infection and Immunity, 79, 4227–4239.10.1128/IAI.05398-11

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