The specificity of ParR binding determines the compatibility of conjugative plasmids inClostridium perfringens

Abstract

19Plasmids that encode the same replication machinery are generally unable to coexist in the same 20 bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or 21 antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many 22 bacteria, plasmid partitioning upon cell division involves a ParMRC system and there are ~10 different 23ParMRC families in C. perfringens, with differences in amino acid sequences between each ParM family 24 (15% -54% identity). Since plasmids encoding genes belonging to the same ParMRC family are not 25 observed in the same strain, these families appear to represent the basis for plasmid compatibility in 26 C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-27 binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site 28 from the same ParMRC family, but could not interact with a parC site from a different ParMRC family. 29These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is 30 mediated by their parMRC-like partitioning systems. This process provides a selective advantage by 31 enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for 32 different host targets. 33 34 35 36 37 38 39 40 41Low-copy number plasmids usually require an active partitioning system to ensure that they are faithfully 42 inherited by daughter cells upon cell division (1). Type II or ParMRC plasmid partitioning systems 43 encode three components: parC, a plasmid-encoded centromere, ParM, an actin-like ATPase that 44 forms filaments in the presence of ATP or GTP and ParR, a DNA-binding adaptor protein that binds to 45 parC (2-6). ParMRC systems stabilise the inheritance of plasmids by positioning them on either side of 46 the cell septum prior to cell division. 47ParR proteins are typically ribbon-helix-helix proteins that bind direct repeats within parC, either as a 48 dimer or a dimer of dimers (5,7-10). The parC centromere usually consists of a series of direct repeats 49 upstream of the parM gene, however, its precise genetic structure differs between plasmids. Binding of 50 ParR acts to seed the formation of a higher order solenoid-shaped structure, termed the segrosome, 51where the DNA wraps around ParR leaving a core of ParM interaction sites (9,10). Polymerising ParM 52 filaments then link the ParR-parC complexes of two sister plasmids and push them to either cell pole 53(2,3,11-13). The initial step, in which ParR recognises and interacts with parC, is important in 54 determining partition specificity between plasmids. 55Plasmid incompatibility generally occurs when two co-resident plasmids encode the same essential 56 replication or partitioning machinery (14). Most studies to date have focused on the partition specificity 57 and incompatibility mediated by Type I or ParABS partitioning systems (15) (16-18), there is only limited 58 evidence that partition...

Pathogenicity and virulence of Clostridium perfringens

“…Bioinformatic and functional studies showed that sequence differences in their partitioning and segregation genes are responsible for this plasmid compatibility [ 124 , 134 ]. Recent studies revealed that C. perfringens tcp -conjugative plasmids may have up to 10 different types of partitioning system (ParMRC A-J ) located in the core region of these plasmids [ 150 , 151 ] and those variations in partitioning machinery enable a single C. perfringens strain to carry multiple discrete plasmids. Another intriguing feature of pCW3-like plasmids is that their replication protein is highly similar and specific to C. perfringens plasmids [ 120 , 121 , 126 ], which may explain why these plasmids have not adapted to other bacterial species.…”