-
Molecular Biology and Evolution Dec 2021Plasmids are extrachromosomal genetic elements in prokaryotes that have been recognized as important drivers of microbial ecology and evolution. Plasmids are found in...
Plasmids are extrachromosomal genetic elements in prokaryotes that have been recognized as important drivers of microbial ecology and evolution. Plasmids are found in multiple copies inside their host cell where independent emergence of mutations may lead to intracellular genetic heterogeneity. The intracellular plasmid diversity is thus subject to changes upon cell division. However, the effect of plasmid segregation on plasmid evolution remains understudied. Here, we show that genetic drift during cell division-segregational drift-leads to the rapid extinction of novel plasmid alleles. We established a novel experimental approach to control plasmid allele frequency at the levels of a single cell and the whole population. Following the dynamics of plasmid alleles in an evolution experiment, we find that the mode of plasmid inheritance-random or clustered-is an important determinant of plasmid allele dynamics. Phylogenetic reconstruction of our model plasmid in clinical isolates furthermore reveals a slow evolutionary rate of plasmid-encoded genes in comparison to chromosomal genes. Our study provides empirical evidence that genetic drift in plasmid evolution occurs at multiple levels: the host cell and the population of hosts. Segregational drift has implications for the evolutionary rate heterogeneity of extrachromosomal genetic elements.
Topics: Chromosomes; Genetic Drift; Phylogeny; Plasmids; Prokaryotic Cells
PubMed: 34550379
DOI: 10.1093/molbev/msab283 -
PloS One 2016Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26... (Comparative Study)
Comparative Study
BACKGROUND
Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.
RESULTS
Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.
CONCLUSION
Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.
Topics: Alphaproteobacteria; Biological Evolution; Chromosomes, Bacterial; DNA, Bacterial; Evolution, Molecular; Gammaproteobacteria; Gene Duplication; Gene Transfer, Horizontal; Genes, Bacterial; Genome, Bacterial; Molecular Sequence Annotation; Phylogeny; Plasmids; Rickettsia; Sequence Alignment; Species Specificity
PubMed: 26866478
DOI: 10.1371/journal.pone.0147492 -
BMC Bioinformatics Apr 2018In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological...
BACKGROUND
In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological conditions. Such intrinsically disordered proteins (IDPs) and intrinsically disordered (protein) regions (IDRs) are involved in essential cell processes through two basic mechanisms: the entropic chain mechanism which is responsible for rapid fluctuations among many alternative conformations, and molecular recognition via short recognition elements that bind to other molecules. IDPs possess a high adaptive potential and there is special interest in investigating their involvement in organism evolution.
RESULTS
We analyzed 2554 Bacterial and 139 Archaeal proteomes, with a total of 8,455,194 proteins for disorder content and its implications for adaptation of organisms, using three disorder predictors and three measures. Along with other findings, we revealed that for all three predictors and all three measures (1) Bacteria exhibit significantly more disorder than Archaea; (2) plasmid-encoded proteins contain considerably more IDRs than proteins encoded on chromosomes (or whole genomes) in both prokaryote superkingdoms; (3) plasmid proteins are significantly more disordered than chromosomal proteins only in the group of proteins with no COG category assigned; (4) antitoxin proteins in comparison to other proteins, are the most disordered (almost double) in both Bacterial and Archaeal proteomes; (5) plasmidal proteins are more disordered than chromosomal proteins in Bacterial antitoxins and toxin-unclassified proteins, but have almost the same disorder content in toxin proteins.
CONCLUSION
Our results suggest that while disorder content depends on genome and proteome characteristics, it is more influenced by functional engagements than by gene location (on chromosome or plasmid).
Topics: Archaea; Archaeal Proteins; Bacteria; Bacterial Proteins; Chromosomes, Archaeal; Chromosomes, Bacterial; Intrinsically Disordered Proteins; Plasmids; Proteome; Toxins, Biological
PubMed: 29699482
DOI: 10.1186/s12859-018-2158-6 -
Frontiers in Cellular and Infection... 2023Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal...
Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.
Topics: Plasmids; Anti-Bacterial Agents; Drug Resistance, Microbial; Conjugation, Genetic; Gene Transfer, Horizontal
PubMed: 37886666
DOI: 10.3389/fcimb.2023.1269732 -
Nature Microbiology Mar 2024Plasmids alter microbial evolution and lifestyles by mobilizing genes that often confer fitness in changing environments across clades. Yet our ecological and...
Plasmids alter microbial evolution and lifestyles by mobilizing genes that often confer fitness in changing environments across clades. Yet our ecological and evolutionary understanding of naturally occurring plasmids is far from complete. Here we developed a machine-learning model, PlasX, which identified 68,350 non-redundant plasmids across human gut metagenomes and organized them into 1,169 evolutionarily cohesive 'plasmid systems' using our sequence containment-aware network-partitioning algorithm, MobMess. Individual plasmids were often country specific, yet most plasmid systems spanned across geographically distinct human populations. Cargo genes in plasmid systems included well-known determinants of fitness, such as antibiotic resistance, but also many others including enzymes involved in the biosynthesis of essential nutrients and modification of transfer RNAs, revealing a wide repertoire of likely fitness determinants in complex environments. Our study introduces computational tools to recognize and organize plasmids, and uncovers the ecological and evolutionary patterns of diverse plasmids in naturally occurring habitats through plasmid systems.
Topics: Humans; Metagenome; Algorithms; Life Style; Machine Learning; Plasmids
PubMed: 38443576
DOI: 10.1038/s41564-024-01610-3 -
Biophysical Journal Mar 2019Bacterial high-copy-number (hcn) plasmids provide an excellent model to study the underlying physical mechanisms of DNA segment segregation in an intracellular context....
Bacterial high-copy-number (hcn) plasmids provide an excellent model to study the underlying physical mechanisms of DNA segment segregation in an intracellular context. Using two-color fluorescent repressor-operator systems and a synthetic repressible replication origin, we tracked the motion and segregation of single hcn plasmid molecules in individual cells. The plasmid diffusion dynamics revealed between-plasmid temporal associations (clustering) as well as entropic and elastic recoiling forces in the confined intracellular spaces outside of nucleoids. These two effects could be effectively used in models to predict the heterogeneity of segregation. Additionally, the motile behaviors of hcn plasmids provide quantitative estimates of entropic exclusion strength and dynamic associations between DNA segments. Overall, this study utilizes a, to our knowledge, novel approach to predict the polymer dynamics of DNA segments in spatially confined, crowded cellular compartments as well as during bacterial chromosome segregation.
Topics: Cell Division; DNA; Movement; Plasmids; Single-Cell Analysis
PubMed: 30773297
DOI: 10.1016/j.bpj.2019.01.019 -
Applied and Environmental Microbiology Aug 2016Enterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance...
UNLABELLED
Enterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer of E. faecalis plasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a "self-sensing signal," reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373.
IMPORTANCE
Enterococcus faecalis is a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.
Topics: Bacterial Proteins; Conjugation, Genetic; Enterococcus faecalis; Gene Expression Regulation, Bacterial; Plasmids; Quorum Sensing
PubMed: 27208137
DOI: 10.1128/AEM.00363-16 -
Estimating plasmid conjugation rates: A new computational tool and a critical comparison of methods.Plasmid May 2022Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they...
Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they spread horizontally via conjugation. Here, we compare commonly used methods on simulated and experimental data, and show that the resulting conjugation rate estimates often depend strongly on the time of measurement, the initial population densities, or the initial ratio of donor to recipient populations. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates. We derive a new 'end-point' measure to estimate conjugation rates, which extends the well-known Simonsen method to include the effects of differences in population growth and conjugation rates from donors and transconjugants. We further derive analytical expressions for the parameter range in which these approximations remain valid. We present an easy to use R package and web interface which implement both new and previously existing methods to estimate conjugation rates. The result is a set of tools and guidelines for accurate and comparable measurement of plasmid conjugation rates.
Topics: Anti-Bacterial Agents; Bacteria; Conjugation, Genetic; Gene Transfer, Horizontal; Plasmids
PubMed: 35271855
DOI: 10.1016/j.plasmid.2022.102627 -
Infection and Immunity Mar 2013Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role...
Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.
Topics: Animals; Bacterial Proteins; Cell Line; Chlamydia trachomatis; Chromosomes, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Mice; Plasmids; Protein Array Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Virulence
PubMed: 23319558
DOI: 10.1128/IAI.01305-12 -
Applied and Environmental Microbiology Apr 2022The genus encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native...
The genus encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several species. Here, we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis IrR/Munich mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the and genes of pRM with various portions of the region surrounding these genes and a selection reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate's Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM and . PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in transformants. Determination of native pRM copy number using a plasmid-carried gene (RM_p5) in comparison to chromosomally carried indicated reduced copy numbers in transformants. In transformed strains, native pRM and shuttle vectors with homologous and formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii, can cause severe illness or death. Plasmids are found in a large percentage of nonpathogenic rickettsiae, but not in species that cause severe disease. Studying these plasmids can reveal their role in the biology of these bacteria, as well as the molecular mechanism whereby they are maintained and replicate in rickettsiae. Here, we describe a new series of shuttle plasmids for the transformation of rickettsiae based on and sequences of plasmid pRM from Rickettsia monacensis. These shuttle vectors support transformation of diverse rickettsiae, including the native host of pRM, and are useful for investigating genetic determinants that govern rickettsial virulence or their ability to function as symbionts.
Topics: Genetic Vectors; Host Specificity; Plasmids; Rickettsia
PubMed: 35323021
DOI: 10.1128/aem.00210-22