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

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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Conjugative transfer of broad host range plasmids to an acidobacterial strain, Edaphobacter aggregans

Journal of Biotechnology ◽

10.1016/j.jbiotec.2016.01.025 ◽

2016 ◽

Vol 221 ◽

pp. 107-113 ◽

Cited By ~ 3

Author(s):

Emna Bouhajja ◽

Theocharis Efthymiopoulos ◽

Isabelle F. George ◽

David Moreels ◽

Rob Van Houdt ◽

...

Keyword(s):

Host Range ◽

Broad Host Range ◽

Conjugative Transfer ◽

Broad Host Range Plasmids

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The R1162 Mob Proteins Can Promote Conjugative Transfer from Cryptic Origins in the Bacterial Chromosome

Journal of Bacteriology ◽

10.1128/jb.01471-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1574-1580 ◽

Cited By ~ 12

Author(s):

Richard Meyer

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Active Site ◽

Bacterial Chromosome ◽

Broad Host Range ◽

Conjugative Transfer ◽

Chromosomal Dna ◽

Bacterial Dna ◽

Pectobacterium Atrosepticum ◽

Broad Host Range Plasmid

ABSTRACT The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in Pectobacterium atrosepticum, is shown here. A second active site was identified in Escherichia coli, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.

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A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species.

Journal of Bacteriology ◽

10.1128/jb.169.5.1899-1904.1987 ◽

1987 ◽

Vol 169 (5) ◽

pp. 1899-1904 ◽

Cited By ~ 20

Author(s):

R Leemans ◽

E Remaut ◽

W Fiers

Keyword(s):

Host Range ◽

Expression Vector ◽

Interleukin 2 ◽

Broad Host Range ◽

Coliphage Lambda

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Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

Journal of Bacteriology ◽

10.1128/jb.183.3.1022-1031.2001 ◽

2001 ◽

Vol 183 (3) ◽

pp. 1022-1031 ◽

Cited By ~ 28

Author(s):

Malgorzata Zatyka ◽

Lewis Bingle ◽

Anthony C. Jones ◽

Christopher M. Thomas

Keyword(s):

Host Range ◽

Pair Formation ◽

Broad Host Range ◽

Conjugative Transfer ◽

Mating Pair ◽

Transcription Start ◽

Broad Host Range Plasmid ◽

Plasmid Genes ◽

High Degree ◽

Plasmid Rk2

ABSTRACT The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer.trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the −10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.

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A Bacterial Expression Vector Archive(BEVA) for flexible modular assembly of Golden Gate-compatible vectors

10.1101/392423 ◽

2018 ◽

Cited By ~ 1

Author(s):

Barney A. Geddes ◽

Marcela A. Mendoza-Suárez ◽

Philip S. Poole

Keyword(s):

Gene Expression ◽

Host Range ◽

Restriction Enzyme ◽

Copy Number ◽

Expression Vector ◽

Assembly System ◽

Broad Host Range ◽

Golden Gate ◽

Modular Assembly ◽

Golden Gate Cloning

ABSTRACTWe present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. To demonstrate the potential of the system three example vectors were constructed and tested. Golden Gate level 1 vectors; pOGG024, with a broad-host range and high copy number was used for gene expression in laboratory-culturedRhizobium leguminosarum, and pOGG026, with a broad-host range a lower copy number and excellent stability, even in the absence of antibiotic selection. The application of pOGG026 is demonstrated in environmental samples by bacterial gene expression in nitrogen-fixing nodules on pea plants roots formed byR. leguminosarum. Finally, the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, for which we demonstrate an application by constructing a dual reporter plasmid expressing green and red fluorescent proteins.IMPORTANCEModular assembly is powerful as it allows easy combining of different components from a library of parts. In designing a modular vector assembly system, the key constituent parts (and modules) are; an origin of plasmid replication, antibiotic resistance marker(s), cloning site(s), together with additional accessory modules as required. In an ideal vector, the size of each module would be minimized, and this we have addressed. We have designed such a vector assembly system by utilizing the Type IIS restriction enzyme Esp3I and have demonstrated its use for Golden Gate cloning inEscherichia coli. An important attribute of this modular vector assembly is that using the principles outlined here, new modules for specific applications, e.g. origin of replication for plasmids in other bacteria, can easily be designed. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.02338-10 ◽

2011 ◽

Vol 77 (7) ◽

pp. 2414-2427 ◽

Cited By ~ 10

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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