Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

Abstract

The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parS RA3 , forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nic operon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding Large, low-copy-number plasmids displaying a broad host range (BHR) carry an extended backbone of operons involved in replication, copy number control, maintenance, and in the case of self-transmissible plasmids, also conjugative transfer. Expression of these genetic units is driven by high-activity promoters and therefore imposes a substantial metabolic cost on the bacterial cell. To minimize the metabolic burden to the host, regulatory networks have evolved to diminish the backbone gene expression to a low basal level while still allowing for rapid upregulation of the transcription when needed. The regulation is achieved by autogenous repressors that produce negative-feedback loops, mediumrange repressors that control particular modules, and globally acting regulators that bind operators scattered along the plasmid molecule to coordinate expression of all modules. Fine-tuning of gene expression is postulated to be achieved by the action of corepressors and their cooperative binding in the promoter regions, which makes the plasmid highly responsive and facilitates its adaptation (1-4).BHR plasmids from the incompatibility group IncU are widespread and ubiquitous in various aquatic environments, freshwater, fish farms, and clinical isolates (5-8). The modular-mosaic backbone of the IncU plasmids is extremely well conserved not only in its overall genetic organization but also at the nucleotide sequence level (8-10). The mosaic character of genomes from this group is demonstrated by homology of the functional blocks involved in replication, stable maintenance, and conjugative transfer to the respective functional modules of plasmids from different incompatibility groups. Thus, the stability region of RA3 encodes seven homologs of IncP-1 proteins (11), whereas the RA3 conjugative transfer region clusters with similarly organized modules from the PromA group of plasmids (12-16). The RA3 plasmid (Fig. 1), the group archetype isolated from the aquatic bacterium Aeromonas hydrophila (17), is the best-studied IncU representative. Its DNA sequence has been established (GenBank accession no. DQ401103) (10), and individual functional modules have been analyzed ...

Regulation of conjugative transfer of plasmids and integrative conjugative elements

“…Transfer of E. coli plasmid F is influenced by the extracellular response element CpxA (Gubbins et al, 2002). In plasmid RA3 from the IncU group, the segregation proteins KorB and KorC repress the mobC-nic operon, which is required for DNA transfer (Kulinska et al, 2016;Ludwiczak et al, 2013). Also, both the virulence plasmid pSLT from S. enterica and the antibiotic resistance plasmid R100, originally isolated from Shigella, are conjugative in response to host-encoded regulators, such as Dam methylation and Lrp protein (Camacho and Casadesus, 2002;Camacho et al, 2005).…”

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

“…However, in the Class Ia type of par loci, ParA acts as a repressor, binding in the promoter region of the parAB operon, whereas ParB usually works as a co-repressor [41,99,100]. The known examples of plasmidic ParBs of Class Ia playing the role of the global transcriptional regulators include the KorBs of IncP-1 plasmids [126,198,199] and related KorB of IncU plasmids [200].…”

Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif

“…In addition to their main function in plasmid partitioning, plasmidic ParBs of class IA may act as global transcriptional regulators by binding to their operators, spreading and non-discriminately silencing nearby promoters, e.g. in the IncP-1 plasmids ( 56 , 57 ) or IncU plasmids ( 58 ). In contrast, oligomers of chromosomal ParBs spread by up to 20 kb from the parS s ( 18–20 , 54 ) under native conditions but seem to affect gene expression very selectively.…”