Functional analysis of replication and stability regions of broad-host-range conjugative plasmid CTX-M3 from the IncL/M incompatibility group

Plasmid ◽  
2007 ◽  
Vol 57 (2) ◽  
pp. 95-107 ◽  
Author(s):  
Jolanta Mierzejewska ◽  
Anna Kulińska ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Functional Analysis ◽  
Host Range ◽  
Conjugative Plasmid ◽  
Broad Host Range ◽  
Incompatibility Group ◽  
Stability Regions

scholarly journals The Centromere Site of the Segregation Cassette of Broad-Host-Range Plasmid RA3 Is Located at the Border of the Maintenance and Conjugative Transfer Modules

2011 ◽  
Vol 77 (7) ◽  
pp. 2414-2427 ◽  
Author(s):  
Anna Kulińska ◽  
Yunhong Cao ◽  
Małgorzata Macioszek ◽  
Finbarr Hayes ◽  
Grażyna Jagura-Burdzy
Keyword(s):  
Host Range ◽  
Transcriptional Control ◽  
Conjugative Plasmid ◽  
Broad Host Range ◽  
Conjugative Transfer ◽  
Incompatibility Group ◽  
Partition System ◽  
Promoter Motif ◽  
Broad Host Range Plasmid ◽  
Low Copy Number

ABSTRACTRA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally fromAeromonasspp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon inEscherichia coli. This fragment specifies thekorA-incC-korB-orf11operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative −35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ∼300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ∼300-bp fragment also encompasses the origin of conjugative transfer,oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set ofcis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

1982 ◽  
Vol 185 (2) ◽  
pp. 223-238 ◽  
Author(s):  
Tobias Kieser ◽  
David A. Hopwood ◽  
Helen M. Wright ◽  
Charles J. Thompson
Keyword(s):  
Functional Analysis ◽  
Host Range ◽  
Broad Host Range ◽  
Cloning Vectors ◽  
Dna Cloning

scholarly journals A Type IV-Secretion-Like System Is Required for Conjugative DNA Transport of Broad-Host-Range Plasmid pIP501 in Gram-Positive Bacteria

2007 ◽  
Vol 189 (6) ◽  
pp. 2487-2496 ◽  
Author(s):  
Mohammad Y. Abajy ◽  
Jolanta Kopeć ◽  
Katarzyna Schiwon ◽  
Michal Burzynski ◽  
Mike Döring ◽  
...  
Keyword(s):  
Host Range ◽  
Sequence Similarity ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Computer Assisted ◽  
Transcriptional Level ◽  
Broad Host Range ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Type Iv

ABSTRACT Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

scholarly journals Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance

2020 ◽  
Vol 87 (2) ◽  
Author(s):  
Ewa Lewicka ◽  
Monika Mitura ◽  
Kamil Steczkiewicz ◽  
Justyna Kieracinska ◽  
Kamila Skrzynska ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Regulatory Function ◽  
Broad Host Range ◽  
Dependent Manner ◽  
Incompatibility Group ◽  
Plasmid Maintenance ◽  
Helical Protein

ABSTRACT KfrA, encoded on the broad-host-range RA3 plasmid, is an alpha-helical DNA-binding protein that acts as a transcriptional autoregulator. The KfrARA3 operator site overlaps the kfrA promoter and is composed of five 9-bp direct repeats (DRs). Here, the biological properties of KfrA were studied using both in vivo and in vitro approaches. Localization of the DNA-binding helix-turn-helix motif (HTH) was mapped to the N29-R52 region by protein structure modeling and confirmed by alanine scanning. KfrA repressor ability depended on the number and orientation of DRs in the operator, as well as the ability of the protein to oligomerize. The long alpha-helical tail from residues 54 to 355 was shown to be involved in self-interactions, whereas the region from residue 54 to 177 was involved in heterodimerization with KfrC, another RA3-encoded alpha-helical protein. KfrA also interacted with the segrosome proteins IncC (ParA) and KorB (ParB), representatives of the class Ia active partition systems. Deletion of the kfr genes from the RA3 stability module decreased the plasmid retention in diverse hosts in a species-dependent manner. The specific interactions of KfrA with DNA are essential not only for the transcriptional regulatory function but also for the accessory role of KfrA in stable plasmid maintenance. IMPORTANCE Alpha-helical coiled-coil KfrA-type proteins are encoded by various broad-host-range low-copy-number conjugative plasmids. The DNA-binding protein KfrA encoded on the RA3 plasmid, a member of the IncU incompatibility group, oligomerizes, forms a complex with another plasmid-encoded, alpha-helical protein, KfrC, and interacts with the segrosome proteins IncC and KorB. The unique mode of KfrA dimer binding to the repetitive operator is required for a KfrA role in the stable maintenance of RA3 plasmid in distinct hosts.

scholarly journals Genome organization of thePseudomonas aeruginosanarrow host range plasmid R91–5 determined by deletion and cloning analysis

1985 ◽  
Vol 45 (2) ◽  
pp. 195-198 ◽  
Author(s):  
R. J. Moore ◽  
V. Krishnapillai
Keyword(s):  
Genetic Analysis ◽  
Host Range ◽  
Restriction Enzyme ◽  
Genome Organization ◽  
Conjugative Plasmid ◽  
Deletion Mutants ◽  
Broad Host Range ◽  
Dna Fragments ◽  
Narrow Host Range ◽  
Fertility Inhibition

SummaryBy physical and genetic analysis of deletion mutants of the narrow host range IncP-10P. aeruginosaconjugative plasmid R91–5 it has been shown that the phenotypes related to its transfer, namely the inhibition of the replication of the phage G101, entry exclusion and the fertility inhibition of the wide host range plasmid R18 map at kilobase coordinates 19·7–20·7, 18·5–19·7, 28·8–30·15 and/or 34·9–36·15, respectively. These locations have been confirmed by cloning restriction enzyme generated DNA fragments of R91–5 into a small broad host range, multicopy non-conjugative plasmid.

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

2016 ◽  
Vol 82 (8) ◽  
pp. 2320-2335 ◽  
Author(s):  
Anna Kulinska ◽  
Jolanta Godziszewska ◽  
Anna Wojciechowska ◽  
Marta Ludwiczak ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Transcriptional Repression ◽  
Conjugative Plasmid ◽  
Broad Host Range ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Binding Factor ◽  
Stable Plasmid ◽  
Stable Maintenance

ABSTRACTThe 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 sequenceparSRA3, 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 adjacentmobCpromoter driving expression of themobC-nicoperon, 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 to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions inEscherichia coli, since its overexpression intransnegatively interferes with mini-RA3 replication and stable maintenance of RA3.

scholarly journals Functional Analysis of Three Plasmids from Lactobacillus plantarum

2005 ◽  
Vol 71 (3) ◽  
pp. 1223-1230 ◽  
Author(s):  
Richard van Kranenburg ◽  
Natasa Golic ◽  
Roger Bongers ◽  
Rob J. Leer ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Host Range ◽  
Lactobacillus Plantarum ◽  
Unknown Function ◽  
Conjugative Plasmid ◽  
Rolling Circle Replication ◽  
Cloning Vectors ◽  
Rolling Circle ◽  
Arsenite Resistance

ABSTRACT Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.

scholarly journals Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

2008 ◽  
Vol 74 (13) ◽  
pp. 4119-4132 ◽  
Author(s):  
Anna Kulinska ◽  
Magdalena Czeredys ◽  
Finbarr Hayes ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Host Range ◽  
Antibiotic Resistance Gene ◽  
Functional Characterization ◽  
Repetitive Sequences ◽  
Replication Initiation ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Broad Host Range ◽  
Conjugative Transfer ◽  
Stable Maintenance

ABSTRACT IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

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