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 IncP Plasmid Carrying Colistin Resistance Gene mcr-1 in Klebsiella pneumoniae from Hospital Sewage

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Feifei Zhao ◽  
Yu Feng ◽  
Xiaoju Lü ◽  
Alan McNally ◽  
Zhiyong Zong
Keyword(s):  
Klebsiella Pneumoniae ◽  
Host Range ◽  
Resistance Gene ◽  
Insertion Sequence ◽  
Sequence Type ◽  
Broad Host Range ◽  
Colistin Resistance ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Hospital Sewage

ABSTRACT A Klebsiella pneumoniae strain of sequence type 313 (ST313) recovered from hospital sewage was found carrying the plasmid-borne colistin resistance gene mcr-1, which was bracketed by two copies of the insertion sequence ISApl1 on a 57-kb self-transmissible IncP-type plasmid of a new IncP-1 clade. The carriage of mcr-1 on a self-transmissible broad-host-range plasmid highlights that mcr-1 has the potential to spread beyond the Enterobacteriaceae family.

scholarly journals Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range PromoterPm

2011 ◽  
Vol 77 (8) ◽  
pp. 2648-2655 ◽  
Author(s):  
Rahmi Lale ◽  
Laila Berg ◽  
Friederike Stüttgen ◽  
Roman Netzer ◽  
Marit Stafsnes ◽  
...  
Keyword(s):  
Host Range ◽  
Untranslated Region ◽  
Micrococcus Luteus ◽  
Background Level ◽  
Broad Host Range ◽  
Continuous Control ◽  
Wild Type ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Genes Encoding

ABSTRACTThe induciblePmpromoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in thePmDNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) inEscherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter,P1(Pantitet), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50carotenoid sarcinaxanthin in an engineered strain ofE. colithat produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing threeMicrococcus luteusderived genes from the promoterPm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

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.

Thermodynamic Properties and DNA Binding of the ParD Protein from the Broad Host-Range Plasmid RK2/RP4 Killing System

1999 ◽  
Vol 380 (12) ◽  
Author(s):  
Monika Oberer ◽  
Helmut Lindner ◽  
Otto Glatter ◽  
Christoph Kratky ◽  
Walter Keller
Keyword(s):  
Dna Binding ◽  
Thermodynamic Properties ◽  
Host Range ◽  
Broad Host Range ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

scholarly journals Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

1998 ◽  
Vol 180 (22) ◽  
pp. 6023-6030 ◽  
Author(s):  
Carla L. Easter ◽  
Helmut Schwab ◽  
Donald R. Helinski
Keyword(s):  
Host Range ◽  
Plasmid Stability ◽  
Broad Host Range ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Resolution System ◽  
Stable Maintenance ◽  
Plasmid Stabilization ◽  
Plasmid Rk2

ABSTRACT The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, andparCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or bothparB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss ofparCBA activity. For P. aeruginosaPAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosaPAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parAand res alone were sufficient for stabilization. Thecer resolvase system of plasmid ColE1 and theloxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all threeE. coli strains. These observations indicate that theparA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of theparC and parB genes but that in at least oneE. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or thecer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

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.

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