DNA-Binding Proteins Regulating pIP501 Transfer and Replication

Abstract

pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently detected in clinical Enterococcus faecalis and Enterococcus faecium strains. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual function: It acts as transcriptional repressor at the repR promoter and, in addition, prevents convergent transcription of RNAIII and repR mRNA (RNAII), which indirectly increases RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including Streptomyces and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter overlapping with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein) and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

“…The monomeric Helix-Turn-Helix protein TraN of the Enterococcus faecalis conjugative plasmid pIP501 binds to its oriT region, which suggested that it might be an auxiliary protein of pIP501. However, recent results revealed that traN is not essential for conjugation, and it is now believed that it may be a repressor of conjugation by regulating either the expression of the conjugation operon or activity of the relaxase TraA ( Goessweiner-Mohr et al, 2014 ; Grohmann et al, 2016 ).…”

“…Due to its crucial role in conjugation, relaxases have attained considerable attention and several of them have been characterized in detail at the biochemical, functional and structural levels. In some cases, for instance ICE Bs1 of Bacillus subtilis and the broad host range conjugative plasmid pIP501, the relaxase is the only protein that is required for processing the DNA ( Kopec et al, 2005 ; Lee and Grossman, 2007 ; Grohmann et al, 2016 ). However, in the majority of cases additional protein(s), encoded either by the CE or the host, bind to the oriT and are involved in processing of the DNA.…”

Type IV Secretion in Gram-Negative and Gram-Positive Bacteria

“…These systems are found in most species of 10 Gramand Gram + bacteria, and conjugative plasmids also exist in a few species of archaea (Wagner et al, 2017). In bacteria, these mobile genetic elements contribute to the spread of fitness traits and, more problematically from a clinical perspective, multiple antibiotic resistances (Christie, 2016;Grohmann et al, 2016). As mentioned earlier, the prototypic systems among Gramspecies include the A. tumefaciens VirB/VirD4 T4SS (Christie, 2016), and the E. coli conjugative plasmids F, R388, and pKM101 (Lawley et al, 2003;Llosa and de la Cruz, 2005;de la Cruz et al, 2010;Frost and Koraimann, 2010;Zechner et al, 2012, Arutyunov andFrost, 2013;Koraimann and Wagner, 2014;Cabezon et al, 2015).…”

“…Currently, the best characterized T4SSs from Gram + bacteria are those encoded by the Enterococcus faecalis sex-pheromone responsive plasmid pCF10 (Li et al, 2012;Clewell et al, 2014;Laverde Gomez et al;Bhatty et al, 2015;Whitaker et al, 2015;Bhatty et al, 2017), C. perfringens plasmid pCW3 (Bantwal et al, 2012;Porter et al, 2012;Wisniewski et al, 2015;2016; Rood, 2017) and broad-host-range plasmid pIP501 originally isolated from S. agalactiae (Arends et al, 2013; Goessweiner-Mohr et al, 2013 a and b; 2014 a and b; Fercher et al, 2016;Grohmann et al, 2016;Kohler et al, 2017;Laverde et al, 2017). The conjugation machines in Gram + species differ from their Gramspecies counterparts mainly by lacking the outer membrane core complex and the VirB11 ATPase.…”

Structural and Molecular Biology of Type IV Secretion Systems

“…Conjugation systems of G + bacteria lack the core complex proteins VirB7, VirB9, VirB10 and the pilins VirB2 and VirB5 but the system is still competent for secretion (Alvarez-Martinez and Christie 2009). Homologues of VirD4, VirB4, VirB1 and of the inner membrane proteins VirB6 and VirB8 have been identified in the G + T4SS (Wisniewski and Rood 2017;Grohmann et al 2016) (Figure 2.1). As for G -, there is an important diversity in the composition of G + T4SSs and some, such as the one encoded by the pIP501 conjugative plasmid, present unique proteins that have no sequence similarity with VirB/D proteins ( Figure 2…”