scholarly journals Comparative analysis of MOBQ4 plasmids demonstrates that MOBQ is a cis-acting-enriched relaxase protein family

2019 ◽  
Author(s):  
M. Pilar Garcillán-Barcia ◽  
Raquel Cuartas-Lanza ◽  
Ana Cuevas ◽  
Fernando de la Cruz
Keyword(s):  
Bacterial Genome ◽  
Donor Cell ◽  
Cis Acting ◽  
Biologically Relevant ◽  
E Coli ◽  
Conjugative Plasmids ◽  
Transfer Genes ◽  
Plasmid Mobilization ◽  
Helper Plasmids ◽  
Origin Of Transfer

ABSTRACTA group of small mobilizable plasmids is increasingly being reported in epidemiology surveys of enterobacteria. Some of them encode colicins, while others are cryptic. All of them encode a relaxase belonging to a previously non-described group of the MOBQ class, MOBQ4. While highly similar in their mobilization module, two families with unrelated replicons can be distinguished, MOBQ41 and MOBQ42. Members of both groups were compatible between them and stably maintained in E. coli. MOBQ4 plasmids were mobilized by conjugation. They contain two transfer genes, mobA coding for the MOBQ4 relaxase and mobC, which was non-essential but enhanced the plasmid mobilization frequency. The origin of transfer was located between these two divergently transcribed mob genes. MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative transmission. No interference in mobilization was observed when both MOBQ41 and MOBQ42 were present in the same donor cell. Remarkably, MOBQ4 relaxases exhibited a cis-acting preference for their oriTs, a feature already observed in other MOBQ plasmids. These findings indicate that MOBQ4 plasmids can efficiently spread among enterobacteria aided by coresident IncI1, IncK and IncL/M plasmids, while ensuring their self-dissemination over highly-related elements.IMPORTANCEPlasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. A group of plasmids, called mobilizable, is able to disseminate aided by helper conjugative plasmids. Here, we studied a group of phylogenetically-related mobilizable plasmids, MOBQ4, commonly found in clinically-relevant enterobacteria, uncovering the helper plasmids responsible for their dissemination. We found that the two plasmid species encompassed in the MOBQ4 group can coexist and transfer orthogonally, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

scholarly journals Interactions between the surface exclusion systems of some F-like plasmids

1974 ◽  
Vol 24 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Neil Willetts ◽  
John Maule
Keyword(s):  
Recipient Cell ◽  
Receptor Site ◽  
Donor Cell ◽  
E Coli ◽  
Transfer Genes ◽  
Donor Cells ◽  
The One ◽  
Sites Of Action ◽  
Surface Exclusion ◽  
Transfer Inhibition

SUMMARYFour surface exclusion systems have been identified amongst a group of F-like plasmids inE. coli: SfxI(F), SfxII(ColV2 and R538-1fin−), SfxIII(ColVBtrpand R1-19) and SfxIV(R100-1 and R136fin−). None of these systems was expressed in stationary phase cells or, except for ColVBtrp, duringfin+transfer inhibition, showing that the surface exclusion gene(s) is usually co-controlled with the transfer genes.Recipient cells carrying two plasmids specifying different surface exclusion systems did not always express both of these: the overall pattern suggested that the four systems and/or their sites of action are related. There was no surface exclusion between donor cells carrying two plasmids determining different surface exclusion systems and recipient cells carrying a plasmid determining either one of these. Our hypothesis to explain this and other results is that surface exclusion prevents interaction between the tip of the pilus on the donor cell and a receptor site on the recipient cell surface. Pili (probably mixed) with two types of tips would be present on cells carrying two different plasmids, the one unresponsive to the single surface exclusion system of the recipient cells allowing transfer of both plasmids.

scholarly journals Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

2021 ◽  
Author(s):  
Eric S Tvedte ◽  
Mark Gasser ◽  
Benjamin C Sparklin ◽  
Jane Michalski ◽  
Carl E Hjelmen ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Hybrid Approach ◽  
Cost Effective ◽  
Fruit Fly ◽  
Drosophila Ananassae ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
E Coli ◽  
Hybrid Approaches ◽  
Long Read

Abstract The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

scholarly journals The Location of Substitutions and Bacterial Genome Arrangements

2020 ◽  
Author(s):  
Daniella F Lato ◽  
G Brian Golding
Keyword(s):  
Sinorhizobium Meliloti ◽  
Bacterial Genome ◽  
Evolutionary Analysis ◽  
Origin Of Replication ◽  
Ancestral Reconstruction ◽  
Molecular Change ◽  
E Coli ◽  
A Genome ◽  
The Impact ◽  
Molecular Evolutionary Analysis

Abstract Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, while substitution rates usually increases with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and distance from the origin of replication is often significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), while others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

scholarly journals SHV-16, a β-Lactamase with a Pentapeptide Duplication in the Omega Loop

2001 ◽  
Vol 45 (9) ◽  
pp. 2480-2485 ◽  
Author(s):  
Corinne Arpin ◽  
Roger Labia ◽  
Catherine Andre ◽  
Cécile Frigo ◽  
Zoubida El Harrif ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Donor Cell ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Resistance Pattern ◽  
E Coli ◽  
Omega Loop ◽  
Low Levels ◽  
Encoding Gene ◽  
Level Resistance

ABSTRACT A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 μg/ml), ticarcillin (MIC of 512 μg/ml), and ceftazidime (MIC of 128 μg/ml) and susceptible to all other β-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum β-lactamase (ESBL). Transconjugants inEscherichia coli were obtained at low levels (10−7 per donor cell) and exhibited a similar β-lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 μg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR usingbla SHV-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla SHV-1 orbla SHV-16 in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.

scholarly journals NikAB- or NikB-Dependent Intracellular Recombination between Tandemly Repeated oriT Sequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors

2003 ◽  
Vol 185 (13) ◽  
pp. 3871-3877 ◽  
Author(s):  
Nobuhisa Furuya ◽  
Teruya Komano
Keyword(s):  
Escherichia Coli ◽  
Dna Transfer ◽  
The Other ◽  
E Coli ◽  
Core Sequence ◽  
Bacterial Plasmid ◽  
Plasmid R64 ◽  
Dna Processing ◽  
Origin Of Transfer

ABSTRACT The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.

scholarly journals Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Anne-Claire Mahérault ◽  
Harry Kemble ◽  
Mélanie Magnan ◽  
Benoit Gachet ◽  
David Roche ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Negative Impact ◽  
Fitness Cost ◽  
M Gene ◽  
Content Type ◽  
E Coli ◽  
Conjugative Plasmids ◽  
K 12

ABSTRACT Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

scholarly journals oriT-Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of theSinorhizobium meliloti pExo Megaplasmid Replicator Region

2000 ◽  
Vol 182 (19) ◽  
pp. 5486-5494 ◽  
Author(s):  
Patrick S. G. Chain ◽  
Ismael Hernandez-Lucas ◽  
Brian Golding ◽  
Turlough M. Finan
Keyword(s):  
Replication Origin ◽  
Sinorhizobium Meliloti ◽  
Region Of Interest ◽  
Origin Of Replication ◽  
Bacterial Genomes ◽  
Site Specific ◽  
E Coli ◽  
Transfer Genes ◽  
In Trans ◽  
Bac Cloning

ABSTRACT We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers theoriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, andrepC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.

scholarly journals A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy

2015 ◽  
Vol 184 ◽  
pp. 425-450 ◽  
Author(s):  
Jacek T. Mika ◽  
Aster Vanhecke ◽  
Peter Dedecker ◽  
Toon Swings ◽  
Jeroen Vangindertael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Bacterial Genome ◽  
Genetically Engineered ◽  
Replication Initiation ◽  
Cell Protein ◽  
Experimental Conditions ◽  
Localization Microscopy ◽  
E Coli ◽  
Copy Numbers

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20–30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA–PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA–PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA–PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA–PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA–PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

scholarly journals Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal cis-Acting Sequences

2001 ◽  
Vol 75 (3) ◽  
pp. 1284-1293 ◽  
Author(s):  
Nathalie Clément ◽  
Bernard Avalosse ◽  
Karim El Bakkouri ◽  
Thierry Velu ◽  
Annick Brandenburger
Keyword(s):  
Virus Production ◽  
Dna Amplification ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Helper Plasmid ◽  
Minute Virus Of Mice ◽  
Coding Sequences ◽  
Cis Acting ◽  
Minute Virus ◽  
Helper Plasmids

ABSTRACT The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimalcis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.

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