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.

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 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.

scholarly journals Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Emily A. Sansevere ◽  
Xiao Luo ◽  
Joo Youn Park ◽  
Sunghyun Yoon ◽  
Keun Seok Seo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Integration Site ◽  
Consensus Sequence ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Site Preference ◽  
Conjugative Transfer ◽  
Content Type ◽  
Integrative Conjugative Elements

ABSTRACT ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus. Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA. The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013. Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013. A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

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.

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

1992 ◽  
Vol 6 (7) ◽  
pp. 907-920 ◽  
Author(s):  
Mostafa Motallebi-Veshareh ◽  
Dietmar Balzer ◽  
Erich Lanka ◽  
Grazyna Jagura-Burdzy ◽  
Christopher M. Thomas
Keyword(s):  
Host Range ◽  
Transfer Functions ◽  
Broad Host Range ◽  
Conjugative Transfer ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

scholarly journals Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

2000 ◽  
Vol 66 (9) ◽  
pp. 3856-3867 ◽  
Author(s):  
Rob Meima ◽  
Mary E. Lidstrom
Keyword(s):  
Escherichia Coli ◽  
Deinococcus Radiodurans ◽  
Shuttle Vector ◽  
General Purpose ◽  
Replication Initiation ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Shift Analysis ◽  
Stable Maintenance ◽  
Overexpression System

ABSTRACT The nucleotide sequence of a 12-kb fragment of the crypticDeinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU andresU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcuspromoter was shown to efficiently express LacZ.

scholarly journals Genome Sequence of the Broad-Host-Range Pseudomonas Phage Φ-S1

2012 ◽  
Vol 86 (18) ◽  
pp. 10239-10239 ◽  
Author(s):  
Sanna Sillankorva ◽  
Andrew M. Kropinski ◽  
Joana Azeredo
Keyword(s):  
Host Range ◽  
Genome Sequence ◽  
Open Reading Frames ◽  
Broad Host Range ◽  
Content Type ◽  
Double Stranded Dna ◽  
The Family ◽  
Reading Frames

The broad-host-range lyticPseudomonasphage Φ-S1 possess a 40,192 bp double-stranded DNA (dsDNA) genome of 47 open reading frames (ORFs) and belongs to the familyPodoviridae, subfamilyAutographivirinae, genusT7likevirus.

scholarly journals Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage

2003 ◽  
Vol 185 (17) ◽  
pp. 5220-5233 ◽  
Author(s):  
Eric S. Miller ◽  
John F. Heidelberg ◽  
Jonathan A. Eisen ◽  
William C. Nelson ◽  
A. Scott Durkin ◽  
...  
Keyword(s):  
Host Range ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Gene Clusters ◽  
Open Reading Frames ◽  
Broad Host Range ◽  
Group I Introns ◽  
Repair Enzymes ◽  
Group I

ABSTRACT The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved.

scholarly journals Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol

2018 ◽  
Vol 18 (1) ◽  
Author(s):  
Simon Heinze ◽  
Petra Kornberger ◽  
Christian Grätz ◽  
Wolfgang H. Schwarz ◽  
Vladimir V. Zverlov ◽  
...  
Keyword(s):  
Host Range ◽  
Expression Vector ◽  
Bacillus Species ◽  
Broad Host Range ◽  
Conjugative Transfer
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