Regulation of Gram-Positive Conjugation

Abstract

Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and best-characterized T4SS is that of the Gram-negative soil bacterium Agrobacterium tumefaciens. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from Enterococcus model plasmids pIP501 and pCF10, Bacillus plasmid pLS1, Clostridium plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICEBs1, ICESt1, ICESt3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer.

Keywords: Gram-positive bacteria; conjugation system; integrative and conjugative element; plasmid; regulation; type IV secretion system.

Figure 1

Conjugation of plasmids and integrative and conjugative elements (ICEs). (A) Upon a signal (internal or external), the relaxase introduces a single-strand (ss) break at the nic-site of the origin of transfer (oriT) in the donor cell and covalently binds the ss-DNA. The coupling protein interacts with the relaxosome (relaxase, accessory factors and ss-plasmid DNA) and brings the relaxase-DNA complex to the mating pair formation (MPF) channel. The ss-DNA (most likely covalently bound by the relaxase) is shuttled to the recipient cell. In parallel, replication processes ensure that both donor and recipient harbor a double-stranded version of the conjugative plasmid. (B) With the aid of an excisionase and an integrase, the integrative and conjugative element (ICE) is excised from the host’s chromosome at attL and attR attachment sites, forming an attP site on the circularized ICE and an attB site on the bacterial chromosome. Processing of the DNA, transport and replication follow a comparable mechanism as described for conjugative plasmids in (A). After successful conjugation and replication, the ICE is again integrated into the host’s chromosome.

Figure 2

Regulation of pCF10 conjugative transfer. (A) While both recipient and donor cell produce the pheromone cCF10 from ccfA on the chromosome, only cells harboring pCF10 secrete the inhibitor iCF10 that is produced from prgQ. prgQ lies upstream of the three cassettes responsible for conjugative transfer: The first cassette codes for the surface adhesins PrgA, PrgB, and PrgC and the regulator PrgU, the second harbors members of the Prg/Pcf conjugation system, and the third is composed of genes for processing factors (including the relaxase). The RRNPP-family protein PrgX is the master regulator and PrgY aids in reducing cCF10 pheromone activity. prgZ codes for a permease that imports both cCF10 and iCF10. (B) The relative ratio between cCF10 and iCF10 increases with a higher proportion of potential recipients present. This leads to increased import of cCF10, which binds to PrgX, thus interfering with its ability to repress the P_Q promoter. Transcription of genes required for conjugative transfer is activated, and conjugation takes place. (C) The cCF10/iCF10 ratio decreases with a lower proportion of potential recipients present, which leads to increased import of the inhibitor peptide iCF10. iCF10-PrgX complexes repress the P_Q promoter, thus downregulating transcription of genes required for conjugative transfer and ultimately inhibiting conjugation.

Figure 3

Structural basis for the regulation of the P_Q promoter. (A) The PrgX protein forms tetramers, which repress transfer gene expression by binding to two adjacent operator sites, one of which overlaps with the P_Q promoter. The tetramer is built as a dimer of dimers with each dimer occupying one operator site with the N-terminal DNA-binding domain, whereas the C-terminal domain accommodates the dimerization and pheromone-binding function. The dimer of apo-PrgX (PDB: 2AXU) is shown in a cartoon presentation with the chains colored in blue and green, respectively. (B) Binding of the pheromone cCF10 (red spheres) leads to a conformational change in the C-terminal region of PrgX (PDB: 2AXZ), alleviating the repression of the P_Q promoter. (C) A surplus of iCF10 (orange spheres), which competes with cCF10 for the same binding site (PDB: 2GRL), reinstates the repressed state of the P_Q promoter.

Figure 4

Regulation of pLS20 conjugative transfer. (A) Key players involved in regulation are encoded on the pLS20 plasmid, including the master regulator Rco_LS20 and the Rap_LS20-Phr_LS20 sensor-pheromone cassette upstream of genes required for conjugation. (B) The master regulator Rco_LS20 represses transcription of transfer genes, thus ultimately inhibiting conjugation. (C) Binding of Rap_LS20 to the master regulator Rco_LS20 leads to conformational changes of Rco_LS20, interfering with transcriptional repression of transfer genes, thus conjugation can take place. (D) Upon a distinct pheromone concentration (the quorum), the pheromone Phr*_LS20 interacts with Rap_LS20 and interferes with its binding to the master regulator Rco_LS20. In consequence, Rco_LS20 represses transcription of transfer genes and inhibits conjugation.

Figure 5

Regulation of ICEBs1 conjugative transfer. (A) ICEBs1 harbors most key players involved in regulation of conjugative processes, including the master-regulator ImmR, the metalloprotease ImmA, and the RapI-PhrI sensor-pheromone cassette. (B) Similar to processes in pLS20, the master-regulator ImmR represses transcription of genes involved in mobilization and transfer, which ultimately leads to inhibition of conjugation. (C) RapI or RecA that is activated by general DNA damage control proteolytic cleavage of ImmR by the metalloprotease ImmA. Thus, transcription is activated and conjugation can take place. (D) Upon a distinct concentration (the quorum), the signal peptide PhrI binds to RapI, interfering with its ability to activate ImmA; thus, ImmR can exert repression of the promoter as described in (A).

Figure 6

TraN and TcpK are small regulatory proteins exhibiting a winged-helix-turn-helix (wHTH) fold motif. (A) TraN is a potent repressor of transcription of the pIP501 tra-operon. The TraN protein forms an internal dimer with one wHTH motif in each the N- and C-terminal domain. The TraN-DNA complex (PDB: 6G1T) is shown in cartoon representation, with TraN colored according to secondary structure (α-helices green, β-strands blue) and the DNA in grey. The view is along the recognition helices inserted in the major grooves of the recognition site (left panel) and along the pseudo-two-fold axis of the TraN molecule (right panel). (B) The TcpK-DNA complex (PDB: 5VFX) contains two protein dimers bridging two double-stranded DNA molecules with two direct-repeat recognition sequences. The color scheme is the same as in panel (A), and the orientation is chosen to show the DNA-binding mode with the β-ribbon of the wing inserted in the major groove of the double-stranded DNA (left panel) and the bridging of the two double-stranded DNA pieces by one of the TcpK dimers (right panel).

Figure 7

Regulation of pIP501 conjugative transfer. (A) Transcription of the pIP501 transfer genes is executed by two promoters, P_tra and P_traNO, leading to the production of factors required for conjugative transfer. (B) By a concerted action between the relaxase TraA and TraN, transcription from the P_tra promoter is reduced. Further, TraN can repress the P_traNO promoter upstream of its own gene. These regulative processes lead to inhibition of conjugative transfer.

Figure 8

Structure of the ArtA-DNA complex (PDB: 3GXQ). ArtA, the master regulator of the plasmid pSK41, exhibits a ribbon-helix-helix fold. The dimeric repressor is shown in cartoon representation, with chains A and B colored in green and blue, respectively. The anti-parallel β-ribbon permeates the major groove of the binding-site DNA and exerts base-specific interactions (left panel). The view along the twofold axis of the ArtA dimer and the pseudo-palindromic double-stranded DNA site is shown in the right panel.