Two distinct conjugal transfer systems on Streptomyces plasmid pZL1.

The mechanism of conjugal transfer of plasmids in Gram-negative and unicellular Gram-positive bacteria is commonly via a type IV secretion system (T4SS) [1]. The genes encoding the T4S proteins are usually arranged in a single operon or a few operons. In Gram-negative Agrobacterium tumefaciens, the T4SS is encoded by the virB and virD operons (11 and 5 genes, respectively) [2]. Streptomyces are multicellular mycelial Gram-positive bacteria that form unicellular spores. There are fundamental differences in the mechanisms of conjugal transfer between Streptomyces plasmids and those of Gram-negative and unicellular Gram-positive bacteria. Conjugal transfer of Streptomyces plasmids requires a tra gene encoding an FtsK/SpoIIIE-family DNA translocator and a few adjacent genes [3]. No bacterial T4SS has previously been found on Streptomyces conjugative plasmids. We reported here the coexistence of both a T4SS-like and an FtsK/SpoIIIE-family DNA translocator on a 128-kb Streptomyces plasmid, pZL1. Using a method of recombinational cloning [4], an 130-kb pZL1/pQX17 co-integrated plasmid (pZQ96) was cloned in Escherichia coli. Shotgun sequencing and assembling of the complete nucleotide sequence of pZQ96 showed that pZL1 is comprised of 127,862 bp, with 70.5% GþC content. Of 127 open reading frames predicted by ‘FramePlot 4.0 beta’ (http://nocardia.nih.go.jp/fp4/), 41 resemble the genes with known function and 86 are hypothetical or unknown genes. Surprisingly, four (i.e. pZL1.38, pZL1.40, pZL1.41, and pZL1.43c) of the nine clustered genes (pZL1.35–pZL1.43c) encoded VirB6, VirB4, VirD4, and TraA, respectively, resembling those of the T4SS components of bacterial conjugative plasmids. TraA (pZL1) is a large protein (1880 amino acids) containing multiple domains including relaxase and helicase, resembling those of the TraI protein (1756 amino acids) of the E. coli F plasmid [identity: 90/281 (32%); expectation value: 2 10]. In another pZL1 locus, a gene (pZL1.95c) encoding an FtsK/SpoIIIE-family translocator resembles the major transfer protein TraB of Streptomyces plasmid pSVH1 [identity: 118/388 (30%); expectation value: 2 10]. These results suggested that two distinct conjugal transfer genes co-existed in plasmid pZL1. To investigate whether the cluster of nine genes (pZL1.35–pZL1.43c) might mediate plasmid conjugal transfer, various fragments were cloned in pQC578 [5], which contained a replication origin of Streptomyces plasmid pSLA2 and a selection marker tsr (thiostrepton resistance) gene. The resulting plasmids (Supplementary Table S1) were introduced by being transformed [6] into Streptomyces lividans ZX7 [7]. These transformants were used as donors. Streptomyces lividans ZX7 containing a chromosomeintegrated pSET152 [8] carrying an aac(3)IV gene, conferring apramycin resistance, was used as a recipient. The donor and recipient strains were co-cultured [9] and spores were plated on the medium containing thiostrepton and/or apramycin. As shown in Fig. 1, plasmid pZLQ201 carrying the intact pZL1.35–pZL1.43c gene cluster could conjugally transfer in S. lividans at a frequency of (3.5+1.6) 10. Deletions of the six genes (i.e. pZL1.35–pZL1.40) in plasmid pZQ205 stimulated about five times of transfer frequency. However, deletions of traA (pZLQ206) or virD4 (pZLQ208,) almost abolished transfer of the plasmids. In comparison to pZLQ201, deletion of a small gene, pZL1.42 (pZLQ207), significantly decreased the transfer frequency ( 100 times). Thus, in the T4SS-like locus on pZL1, only three genes (virD4, pZL1.42, and traA) were essential for plasmid conjugal transfer in S. lividans. To investigate if traB-like pZL1.95c and adjacent genes could mediate plasmid conjugal transfer in S. lividans, various fragments were cloned in pQC578. Streptomyces lividans containing the resulting plasmids were used as donors (Supplementary Table S1), while the same recipient strain as above was used. As shown in Fig. 2, plasmids pZLQ101 and pZLQ102 containing a cluster of eight genes (pZL1.94c– pZL1.101) were required for plasmid transfer at high frequencies ( 5 10) in S. lividans. Deletion of the pZL1.95c gene (designated traB) in pZLQ105 almost abolished the transfer of plasmid ( 3 10). Deletion of pZL1.94c in pZLQ104 decreased transfer frequencies ( 8 10). Acta Biochim Biophys Sin 2014, 46: 1084–1086 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu095. Advance Access Publication 14 October 2014