scholarly journals Experimental Evolution of the Megaplasmid pMPPla107 in Pseudomonas stutzeri Enables Identification of Genes Contributing to Sensitivity to an Inhibitory Agent

2019 ◽  
Author(s):  
Brian A. Smith ◽  
Kevin Dougherty ◽  
Meara Clark ◽  
David A. Baltrus
Keyword(s):  
Pseudomonas Syringae ◽  
Experimental Evolution ◽  
Genetic Basis ◽  
Pseudomonas Stutzeri ◽  
Large Deletion ◽  
Host Cells ◽  
Standard Laboratory ◽  
Fitness Costs ◽  
Killing Activity ◽  
Compensatory Mutations

ABSTRACTHorizontally transferred elements such as plasmids can, at times, burden host cells with various metabolic and fitness costs. Our previous work demonstrated that acquisition of the Pseudomonas syringae megaplasmid pMPPla107 causes sensitivity to a growth inhibiting substance that is produced in cultures during growth under standard laboratory conditions. After 500 generations of laboratory passage of P. stutzeri lines containing pMPPla107, two out of six independent lines displayed resistance to this inhibitory agent. We therefore sequenced the genomes of isolates from each independent evolutionary line to identify the genetic basis of this resistance phenotype through comparative genomics. Our analysis demonstrates that two different compensatory mutations on the megaplasmid ameliorate the sensitivity phenotype: 1) a large deletion of approximately 368kb in pMPPla107 and 2) a SNP in the gene we name skaA for Supernatant Killing Activity. These results provide further evidence that costs associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes.

scholarly journals Experimental evolution of the megaplasmid pMPPla107 in Pseudomonas stutzeri enables identification of genes contributing to sensitivity to an inhibitory agent

2021 ◽  
Vol 377 (1842) ◽  
Author(s):  
Brian A. Smith ◽  
Kevin Dougherty ◽  
Meara Clark ◽  
David A. Baltrus
Keyword(s):  
Pseudomonas Syringae ◽  
Experimental Evolution ◽  
Genetic Basis ◽  
Pseudomonas Stutzeri ◽  
Large Deletion ◽  
Host Cells ◽  
Single Amino Acid ◽  
Fitness Costs ◽  
Nonsynonymous Change ◽  
Single Amino Acid Change

Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the Pseudomonas syringae megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of Pseudomonas stutzeri populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

scholarly journals Plasmid fitness costs are caused by specific genetic conflicts

2021 ◽  
Author(s):  
James Peter John Hall ◽  
Rosanna C. T. Wright ◽  
Ellie Harrison ◽  
Katie J. Muddiman ◽  
Jamie Wood ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Reverse Genetics ◽  
Bacterial Genome ◽  
Fitness Costs ◽  
Generic Properties ◽  
Bacterial Genomes ◽  
Large Plasmids ◽  
Compensatory Mutations ◽  
Genetic Conflicts

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid acquisition are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or expression level. Here we show -- using a combination of experimental evolution, reverse genetics, and transcriptomics -- that fitness costs of two divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid acquisition. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by upregulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained upregulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer due to their propensity for amelioration by single compensatory mutations, explaining why plasmids are so common in bacterial genomes.

scholarly journals Comparative Analysis of the Core Proteomes among the Pseudomonas Major Evolutionary Groups Reveals Species-Specific Adaptations for Pseudomonas aeruginosa and Pseudomonas chlororaphis

Diversity ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 289
Author(s):  
Marios Nikolaidis ◽  
Dimitris Mossialos ◽  
Stephen G. Oliver ◽  
Grigorios D. Amoutzias
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pseudomonas Syringae ◽  
Pseudomonas Stutzeri ◽  
Phylogenomic Analysis ◽  
Pseudomonas Chlororaphis ◽  
Phylogenetic Groups ◽  
The Core ◽  
Core Proteins ◽  
Core Proteome ◽  
Species Specific

The Pseudomonas genus includes many species living in diverse environments and hosts. It is important to understand which are the major evolutionary groups and what are the genomic/proteomic components they have in common or are unique. Towards this goal, we analyzed 494 complete Pseudomonas proteomes and identified 297 core-orthologues. The subsequent phylogenomic analysis revealed two well-defined species (Pseudomonas aeruginosa and Pseudomonas chlororaphis) and four wider phylogenetic groups (Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas putida) with a sufficient number of proteomes. As expected, the genus-level core proteome was highly enriched for proteins involved in metabolism, translation, and transcription. In addition, between 39–70% of the core proteins in each group had a significant presence in each of all the other groups. Group-specific core proteins were also identified, with P. aeruginosa having the highest number of these and P. fluorescens having none. We identified several P. aeruginosa-specific core proteins (such as CntL, CntM, PlcB, Acp1, MucE, SrfA, Tse1, Tsi2, Tse3, and EsrC) that are known to play an important role in its pathogenicity. Finally, a holin family bacteriocin and a mitomycin-like biosynthetic protein were found to be core-specific for P. cholororaphis and we hypothesize that these proteins may confer a competitive advantage against other root-colonizers.

Genetic basis of copper-tolerance in Australian Pseudomonas syringae pv. tomato

2019 ◽  
Vol 48 (4) ◽  
pp. 425-437
Author(s):  
Karina Griffin ◽  
P. Campbell ◽  
C. Gambley
Keyword(s):  
Pseudomonas Syringae ◽  
Genetic Basis ◽  
Copper Tolerance

The coevolution of host resistance and parasitoid virulence

Parasitology ◽  
1998 ◽  
Vol 116 (S1) ◽  
pp. S29-S45 ◽  
Author(s):  
A. R. Kraaijeveld ◽  
J. J. M. Van Alphen ◽  
H. C. J. Godfray
Keyword(s):  
Genetic Variation ◽  
Artificial Selection ◽  
Host Resistance ◽  
Genetic Basis ◽  
Isofemale Line ◽  
Fitness Costs ◽  
Geographical Patterns ◽  
Wide Range ◽  
Great Scope ◽  
Fertile Area

SummaryHost-parasitoid interactions are abundant in nature and offer great scope for the study of coevolution. A particularly fertile area is the interaction between internal feeding parasitoids and their hosts. Hosts have evolved a variety of means of combating parasitoids, in particular cellular encapsulation, while parasitoids have evolved a wide range of countermeasures. Studies of the evolution of host resistance and parasitoid virulence are reviewed, with an emphasis on work involvingDrosophilaand its parasitoids. Genetic variation in both traits has been demonstrated using isofemale line and artificial selection techniques. Recent studies have investigated the fitness costs of maintaining the ability to resist parasitoids, the comparative fitness of flies that have successfully defended themselves against parasitoids, and the degree to which resistance and virulence act against one or more species of host or parasitoid. A number of studies have examined geographical patterns, and sought to look for local adaptation; or have compared the traits across a range of species. Finally, the physiological and genetic basis of change in resistance and virulence is being investigated. While concentrating onDrosophila, the limited amount of work on different systems is reviewed, and other possible areas of coevolution in host-parasitoid interactions are briefly discussed.

scholarly journals Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections

2019 ◽  
Vol 116 (40) ◽  
pp. 20135-20140 ◽  
Author(s):  
Romain Guérillot ◽  
Xenia Kostoulias ◽  
Liam Donovan ◽  
Lucy Li ◽  
Glen P. Carter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Basis ◽  
Host Cells ◽  
Type I ◽  
Modification System ◽  
Persistent Infections ◽  
Restriction Modification System ◽  
Long Read ◽  
Small Colony ◽  
Restriction Modification

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

scholarly journals Inactivation of Genes by Frameshift Mutations Provides Rapid Adaptation of an Attenuated Vaccinia Virus

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Tatiana G. Senkevich ◽  
Erik K. Zhivkoplias ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Experimental Evolution ◽  
Copy Number ◽  
Gene Copy Number ◽  
Myxoma Virus ◽  
Host Cells ◽  
Gene Copy ◽  
Frameshift Mutations ◽  
Adaptive Mutations ◽  
Rapid Acquisition

ABSTRACT Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios. IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.

scholarly journals Isolation, Characterisation and Experimental Evolution of Phage that Infect the Horse Chestnut Tree Pathogen, Pseudomonas syringae pv. aesculi

2020 ◽  
Vol 77 (8) ◽  
pp. 1438-1447
Author(s):  
Sarah L. James ◽  
Mojgan Rabiey ◽  
Benjamin W. Neuman ◽  
Glynn Percival ◽  
Robert W. Jackson
Keyword(s):  
Pseudomonas Syringae ◽  
Experimental Evolution ◽  
Horse Chestnut ◽  
Chestnut Tree

scholarly journals Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release

2020 ◽  
Author(s):  
Juan-Vicente Bou ◽  
Rafael Sanjuán
Keyword(s):  
Experimental Evolution ◽  
Genetic Basis ◽  
Close Association ◽  
Site Directed Mutagenesis ◽  
Full Genome ◽  
Virus Release ◽  
Viral Shedding ◽  
Free Virion ◽  
Viral Particles ◽  
Evolution Experiment

Abstract Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.

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