A novel DNA primase-helicase pair encoded by SCCmec elements

Abstract

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here we biochemically characterize 3 proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3'-to-5' helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec.

How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA

“…Pairwise distance and relatedness ( Figure 1 ) was calculated, revealing a total difference of 10 SNPs (excluding phage regions) and the lack of a 16 kb sequence downstream of the SCC mec integration site ( rlm H) in IMT38951_42 ( Figure 2 ). A closer inspection of the region revealed the loss of a complete SCC mec Vc element ( Figure 2 ) comprising the ccr C8 type recombinase, the C2 mec complex and a recently described putative operon containing an A-family DNA polymerase (CCPol) together with a small protein lacking conserved domains (“middle protein,” MP) as well as a putative helicase (Cch2; referred to as “CCPol-MP-Cch2” I & II in Figures 2 , 3 ), as recently described by Bebel et al (2020) . The region downstream of rlm H in IMT38951 includes a notable repertoire of homologues sequences, i.e., two distinct but closely related ccr C variants and two nearly homologues intergenic spacer regions of 224 bp length, four transposases ( tnp ) of insertion sequence (IS) element 431 (IS431) and two variants of the replication machinery CCPol-MP-Cch2 ( Figure 2 ; Supplementary Table 2 ) (region rlm H – dus , highlighted in green).…”

“…Since sequence identities of genes and intergenic spacer regions present in this region range from 98/99% (light blue-green) to 100% (blue-green), including sequences and genes that flank (regions A and C) the missing SCC mec element in IMT38951_42. DR, direct repeat of 15 bp length; CCPol-MP-Cch2, operon containing an A-family DNA polymerase (CCPol) together with a small protein lacking conserved domains (“middle protein,” MP) as well as a putative helicase (Cch2) ( Bebel et al, 2020 ); ccr C, chromosomal cassette recombinase C; tpn , transposase associated with insertion element (IS) 431; ISR, intergenic spacer region of 224 bp length.…”

“…(C) Both recombination events would allow deletion of an identical and complete SCC mec Vc element. (D) Circular SCC mec element with putative functional recombinase ccr C8 and a CCPol-MP-Cch2 II complex ( Bebel et al, 2020 ) conferring its autonomous replication capabilities. ccr , chromosomal cassette recombinase; IS, insertion sequence; DR, directs repeats associated with ccr -mediated integration and excision of SCC mec elements ( Semmler et al, 2016 ).…”

“…Further sequence comparison with the type V SCC mec (GenBank: AB512767) genes and intergenic regions of a strain (TSGH17) belonging to another livestock-associated genetic background (ST59), used by Bebel et al (2020) to describe the CCPol-MP complex and their Cch2 helicases, revealed a sequence identity of 94.9% and 80 SNPs, two larger insertions/deletions of 40 bp and 1,275 bases with 4 single insertions/deletions proving continuous evolvement of the region downstream of rlm H.…”

“…A further study identified recombination between the putative primase-encoding (now: helicase Cch2, according to Bebel et al, 2020 ) genes belonging to the CCPol-MP-Cch2 complex in the ST398 lineage ( Gómez et al, 2020 ). Other authors have linked the loss of SCC mec with recombination between ccr C allelic variants ( Chlebowicz et al, 2010 ; Vandendriessche et al, 2014 ), a possibility that cannot be ruled out for the isogenic pair belonging to ST398 in our study ( Figure 3 , recombination between B and C).…”