Two novel membrane proteins, TcpD and TcpE, are essential for conjugative transfer of pCW3 in Clostridium perfringens

Abstract

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.

FIG 1

Conjugation frequencies of pCW3 mutants and the respective complemented derivatives. (A) Region of the tcp locus spanning tcpC to tcpF from the wild type (pCW3) and the pCW3ΔtcpD::erm(Q) and pCW3ΔtcpE::erm(Q) mutants. In the mutants, most of the tcpD or tcpE gene has been replaced with the erm(Q) gene. (B) Conjugation frequencies of the pCW3ΔtcpD, pCW3ΔtcpE, and pCW3ΔtcpJ mutants and their respective complementation derivatives. Donors shown on the x axis are isogenic strains in a JIR325 background. pCW3, wild-type pCW3 (JIR4195); ΔtcpJ, pCW3ΔtcpJ (JIR12216); ΔtcpD, pCW3ΔtcpD (JIR12212); ΔtcpD(V), JIR12212 with a vector plasmid (pJIR3422); ΔtcpD(tcpD⁺), JIR12212 with a vector plasmid carrying the wild-type tcpD gene (pJIR4203); ΔtcpD(tcpD-G₆-HA), JIR12212 with a vector plasmid carrying the tcpD HA-tagged derivative (pJIR4204); ΔtcpE, pCW3ΔtcpE (JIR12215); ΔtcpE(V), JIR122125 with a vector plasmid (pJIR3422); ΔtcpE(tcpE⁺), JIR12215 with a vector plasmid carrying the wild-type tcpE gene (pJIR3614); ΔtcpE(tcpE-HA), JIR12215 with a vector plasmid carrying the tcpE HA-tagged derivative (pJIR3757). The transfer frequency is expressed as the number of transconjugants per donor cell. The means ± standard errors of the means are shown, based on results from at least three biological replicates. Statistical analysis was carried out by using an unpaired t test. One asterisk denotes statistical significance (P < 0.05) compared to wild-type pCW3 (positive control). Two asterisks denote statistical significance (P < 0.05) when complemented strains were compared to the respective vector control (V) strain.

FIG 2

Localization of TcpD and TcpE HA-tagged derivatives in C. perfringens cells. Cell fractionation and Western blotting using HA antibodies were carried out on strains expressing either the TcpD or TcpE HA-tagged derivative in a pCW3-positive (+) or pCW3-negative (−) background. pTcpE-HA denotes the vector plasmid carrying the tcpE HA-tagged derivative (pJIR3757), pTcpD-G₆-HA denotes the vector plasmid carrying the tcpD HA-tagged derivative (pJIR4204), and pTcpD-G₆-HA(R) denotes the vector plasmid carrying the tcpD HA-tagged derivative (pJIR4204) rescued from a pCW3-negative background. Expression was examined in a JIR325(pCW3)-positive background in the pCW3ΔtcpE (JIR12215) mutant or the pCW3ΔtcpD (JIR12212) mutant or in a pCW3-negative background in JIR325. The fractions are labeled as follows: L, cell lysate; S, soluble fraction; I, insoluble cell membrane fraction.

FIG 3

Localization of TcpD HA-tagged derivatives in C. perfringens tcp mutant backgrounds. Cell fractionation and Western blotting using HA antibodies were carried out on isogenic derivatives of C. perfringens strain JIR325 expressing the TcpD HA-tagged derivative (pTcpD-G₆-HA) in a pCW3-positive or pCW3-negative background as well as in tcp mutant backgrounds. pTcpD-G₆-HA denotes the vector plasmid carrying the tcpD HA-tagged derivative (pJIR4204) expressed in JIR325 (−) and isogenic strains carrying pCW3 derivatives, abbreviated as follows: ΔD, pCW3ΔtcpD (JIR12212); intP::TT, pCW3intP::TT (JIR12540); ΔA, pCW3ΔtcpA (JIR10251); ΔC, pCW3ΔtcpC (JIR12088); ΔE, pCW3ΔtcpE (JIR12215); ΔF, pCW3ΔtcpF (JIR4940); ΔG, pCW3ΔtcpG (JIR12128); ΔH, pCW3ΔtcpH (JIR4885). The fractions are labeled as follows: L, cell lysate; S, soluble fraction; I, insoluble cell membrane fraction.

FIG 4

Localization of TcpD and TcpE HA-tagged derivatives in C. perfringens. TcpD and TcpE localization was detected in the pCW3 tcpD and tcpE mutant strains complemented with the respective wild-type or HA-tagged derivatives. C. perfringens cells were labeled by using monoclonal anti-HA mouse primary antibodies, followed by Alexa Fluor 488–anti-mouse goat secondary antibodies (green) as well as the nuclear stain DAPI (blue). TcpD, pCW3ΔtcpD background (JIR12212) with a vector plasmid carrying the wild-type tcpD gene (pJIR4203); TcpD-G₆-HA, pCW3ΔtcpD background (JIR12212) with a vector plasmid carrying the tcpD HA-tagged derivative (pJIR4204); TcpE, pCW3ΔtcpE background (JIR12215) with a vector plasmid carrying the wild-type tcpE gene (pJIR3614); TcpE-HA, pCW3ΔtcpE background (JIR12215) with a vector plasmid carrying the tcpE HA-tagged derivative (pJIR3757).