Characterization of the Partitioning System of Myxococcus Plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics

Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

ParAWalker ATPases form part of the machinery that promotes better-thanrandom segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts.IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATPdependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus,the NTD may modulate the polymerisation interface, facilitating polymerisation/ depolymerisation and DNA binding, to promote the cycle that drives partitioning

Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes.

The KorB protein of broad-host-range plasmid RK2 is a transcriptional repressor involved in the control of genes for plasmid replication, conjugative transfer and stable maintenance. We have purified this protein close to homogeneity from cells harbouring an overexpression vector with the korB gene under the control of the tac promoter. KorB binds to restriction fragments bearing its proposed operator sequence, OB. Its interaction with this palindromic site was confirmed by DNaseI or hydroxyl radical footprinting at two OB sequences from RK2. Comparisons showed that the OB context affects the nature of the footprint. Our evidence suggests that KorB is a tetramer. As such, it may be able to bind two sites simultaneously on the same or on different DNA molecules. Using the korABF promoter, which is subject to KorB repression, we demonstrate by footprinting and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprinting showed that KorB represses this promoter by preventing isomerization of the RNA polymerase-promoter complex from the closed to open form

Global transcriptional regulator KorC coordinates expression of three backbone modules of the broad-host-range RA3 plasmid from IncU incompatibility group.

The broad-host-range conjugative RA3 plasmid from IncU incompatibility group has been isolated from the fish pathogen Aeromonas hydrophila. DNA sequencing has revealed a mosaic modular structure of RA3 with the stabilization module showing some similarity to IncP-1 genes and the conjugative transfer module highly similar to that from PromA plasmids. The integrity of the mosaic plasmid genome seems to be specified by its regulatory network. In this paper the transcriptional regulator KorC was analyzed. KorCRA3 (98 amino acids) is encoded in the stabilization region and represses four strong promoters by binding to a conserved palindrome sequence, designated OC on the basis of homology to the KorC operator sequences in IncP-1 plasmids. Two of the KorCRA3-regulated promoters precede the first two cistrons in the stabilization module, one fires towards replication module, remaining one controls a tricistronic operon, whose products are involved in the conjugative transfer process. Despite the similarity between the binding sites in IncU and IncP-1 plasmids, no cross-reactivity between their KorC proteins has been detected. KorC emerges as a global regulator of RA3, coordinating all its backbone functions: replication, stable maintenance and conjugative transfer