The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif

Abstract

Conjugation is a driving force in the evolution and shaping of bacterial genomes. In antibiotic producing streptomycetes even small plasmids replicating via the rolling-circle mechanism are conjugative. Although they encode only genes involved in replication and transfer, the molecular function of most plasmid encoded proteins is unknown. In this work we show that the conjugative plasmid pIJ101 encodes an overlooked protein, SpdA2. We show that SpdA2 is a DNA binding protein which specifically recognizes a palindromic DNA sequence (sps). sps is localized within the spdA2 coding region and highly conserved in many Streptomyces plasmids. Elimination of the palindrome or deletion of spdA2 in plasmid pIJ303 did not interfere with conjugative plasmid transfer or pock formation, but affected segregational stability.

The conjugative DNA-transfer apparatus of Streptomyces

“…SpdA2 of pIJ101 was shown to affect segregational stability (Kieser et al, 2000;Thoma et al, 2014). In contrast, a role of SpdA in plasmid spreading was reported for plasmid pSN22, whereas inactivation of spdA in pJV1 did not cause a phenotype (Kataoka et al, 1991a,b;Servín-González et al, 1995).…”

“…The 120-180 aa proteins contain, with few exceptions, a sequence motif (SRDD/E-X 6-8 -SRDD/E) which coincides with a 12-bp palindromic DNA sequence motif CGTCACGTGACG-N 18-24 -CGTCACGTGACG (Reuther et al, 2006b;Servín-González et al, 1995). The SpdA homologue SpdA2 of pIJ101 was shown to specifically bind to the palindromic sequence motif (Thoma et al, 2014). Bacterial two-hybrid analyses of SpdA of pSVH1 identified SpdB3, Spd79, SpdB2 and TraB as interaction partners.…”

“…Rep, TraR, TraB and SpdA have a specific DNA binding activity and recognize distinct target sequences on the plasmid. Rep binds to the double-stranded origin dso, TraB to clt, TraR to 12 bp repeats (tre boxes) in the promoter region of the tra/spd operon (Kataoka et al, 1994) and SpdA to a palindromic sequence within its own coding region (Thoma et al, 2014). SpdB2 only bound non-specifically to double-stranded DNA suggesting that this protein contributes to a DNA-translocation channel and does not directly recruit a specific DNA substrate.…”

Fluorescence microscopy of Streptomyces conjugation suggests DNA‐transfer at the lateral walls and reveals the spreading of the plasmid in the recipient mycelium

“…Mutations that reduced the pock size and the transconjugant zone, but did not affect the efficiency of transfer from the original donor to the recipient revealed the existence of three to five spd genes on conjugative Streptomyces plasmids (Kieser et al ., ; Kataoka et al ., ; Servín‐González et al ., ). The predicted Spd proteins do not show sequence similarity to any functionally characterized protein and only a few Spd proteins have been biochemically characterized (Pettis et al ., ; Tiffert et al ., ; Thoma et al ., 2014; 2015; Thoma and Muth, ). Whereas the role of TraB in plasmid transfer from the donor into the recipient has been well characterized (Pettis and Cohen, 1994; 1996; 2000; Reuther et al ., ; Vogelmann et al ., ), the molecular mechanism of plasmid spreading in the recipient mycelium has not been elucidated yet and it is unclear whether the FtsK‐like DNA‐translocase TraB is involved or if the Spd proteins can promote the spreading of the plasmid by an TraB‐independent process.…”

A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

“…The role of SpdA in plasmid spreading is unclear. Whereas spdA of plasmid pSN22 has been shown to influence plasmid spreading, the spdA homologue spdA2 of plasmid pIJ101 did not affect pock formation but was characterized as a stability function ( 23 , 24 ). Purified SpdA2 bound to a highly conserved palindromic sequence motif associated with the spdA coding regions of Streptomyces plasmids ( 23 ).…”