Linear plasmids in Klebsiella and other Enterobacteriaceae

Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function, however each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Evidence for a billion-years arms race between viruses, virophages and eukaryotes

The PRD1-adenovirus lineage is one of the oldest and most diverse lineages of viruses. In eukaryotes, they have diversified to an unprecedented extent giving rise to adenoviruses, virophages, Mavericks, Polinton-like viruses and Nucleocytoplasmic Large DNA viruses (NCLDVs) which include the poxviruses, asfarviruses and iridoviruses, among others. Two major hypotheses for their origins have been proposed: the 'virophage first' and 'nuclear escape' hypotheses, but their plausibility until now has remained unexplored. Here, we use maximum-likelihood and Bayesian hypothesis-testing to compare the two scenarios based on the shared proteins forming the virus particle and a comprehensive genomic character matrix. We also compare the phylogenetic origin of the transcriptional proteins shared by NCLDVs and cytoplasmic linear plasmids. Our analyses overwhelmingly favour the virophage first model. These findings shed light on one of the earliest diversifications seen in the virosphere, supporting a billion-years arms race between viruses, virophages and eukaryotes.

Genetic Diversity and The Presence of Circular Plasmids in Bacillus Cereus Isolates of Clinical and Food Origin

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from foods, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed.

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

ABSTRACTEstablishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen’s biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function inCandida albicans

ABSTRACTThe ability to generate autonomously replicating plasmids has been elusive inCandida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively).Candida albicansURA3and a heterologous marker,ARG4,yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologousLEU2marker was highly dependent upon the addition of abona fideorigin sequence. Severalbona fidechromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin,panARS, fromKluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus,C. albicansmaintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.IMPORTANCECircular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, inCandida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate inC. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of aC. albicansorigin and an origin sequence from a distantly related yeast can both function as origins inC. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ betweenC. albicansand baker’s yeast.

Autonomously replicating linear plasmids facilitate the analysis of replication origin function inCandida albicans

The ability to generate autonomously replicating plasmids has been elusive inCandida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences.Furthermore, we could distinguish by colony size, transient, autonomously replicating and chromosomally integrated transformants (tiny, medium and large, respectively).Candida albicans URA3and a heterologous marker,ARG4,yielded many transient transformants indicative of weak origin activity; replication of plasmid carrying heterologousLEU2marker was highly dependent upon the addition of abona fideorigin sequence. Severalbona fidechromosomal origins, with an origin fragment of ~100 bp as well as a heterologous origin,panARS, fromKluyveromyces lactisdrove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus,C. albicansmaintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.ImportanceCircular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, inCandida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate inC. albicans.We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that a ~100 bp fragment of aC. albicansorigin, as well as an origin sequence from a distantly related yeast, can both function as origins inC. albicans.Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ betweenC. albicansand baker’s yeast.

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids fromKluyveromyces lactisas a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5’ and 3’ RACE analysis of all the pGKL1,2 mRNAs and found them not 3’ polyadenylated and containing mostly uncapped 5’ poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5’ capping and/or 5’ polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence thatK2ORF3encodes an mRNA cap guanine-N7-methyltransferase and that 5’ capped pGKL1,2 transcripts contain N7-methylated caps. Translation of pGKL1,2 transcripts is enhanced inIsm1Δandpab1Δstrains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5’ mRNA capping, degree of 5’ non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses.

Complete Genome Sequences of pLMA1 and pLMA7, Two Large Linear Plasmids of Micrococcus Strains Isolated from a High-Altitude Lake in Argentina

ABSTRACT The two linear plasmids pLMA1 (109,112 bp) and pLMA7 (82,075 bp) from Micrococcus strains were isolated from a high-altitude lake in the Argentinean Puna, sequenced, and annotated. These extrachromosomal elements are probably conjugative and harbor genes potentially involved in coping with the harsh conditions in such extreme environments.