Plasmid-free Cells Research Articles

Isolation of an Escherichia coli mutant susceptible to a quinolone in an anaerobic environment

Quinolones are bactericidal agents that interfere with the essential prokaryotic enzyme DNA gyrase. While their mechanism of killing appears to be elucidated, one interesting feature is represented by the fact that, under anaerobic conditions, the growth of bacteria is inhibited but their viability is not affected by the first generation of quinolones such as nalidixic acid. More information about the mode of action of these drugs in anaerobiosis might be gained through the availability of strains subjected to enhanced killing in oxygen-deprived media. It has been assumed that when a population of a AB1157(F’lac) strain is exposed to nalidixic acid, plasmid-free cells could be recovered from culture treated with sub-inhibitory concentrations of the drug (2 mg/L) in aerobiosis, and, at the same drug level, only from the rare spontaneous susceptible mutant(s) in anaerobiosis. Among plasmid free bacteria found, 1 isolate demonstrated the same MIC value to nalidixic acid in both aerobic and anaerobic conditions. The mutation was co-transferred with Tn10 inserted at 28.5 min of the Escherichia coli genetic map into a wildtype strain. These transductants revealed the same phenotypes of the original mutant: susceptibility to nalidixic acid under anaerobic conditions (assessed by time-kill tests) and elongated cells during the aerobic growth, generation time about 65 min in comparison to 25 min of the control. Time kill experiment under aerobic environment revealed that the transductant was also susceptible to ciprofloxacin but not nalidixic acid in the presence of chloramphenicol (50 mg/L). These results suggest a possible role of bacterial topoisomerase in the anaerobic susceptibility to nalidixic acid of the mutant.

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system.

Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new "Ti/Ri eviction plasmids," each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

Phenotypic indications of FtsZ inhibition in hok/sok-induced bacterial growth changes and stress response

The hok/sok locus has been shown to enhance the growth of bacteria in adverse growth conditions such as high temperature, low starting-culture densities and antibiotic treatment. This is in addition to their well-established plasmid-stabilization effect via post-segregational killing of plasmid-free daughter cells. It delays the onset of growth by prolonging the lag phase of bacterial culture, and increases the rate of exponential growth when growth eventually begins. This enables the cells adapt to the prevailing growth conditions and enhance their survival in stressful conditions. These effects functionally complement defective SOS response mechanism, and appear analogous to the growth effects of FtsZ in the SOS pathway. In this study, the role of FtsZ in the hok/sok-induced changes in bacterial growth and cell division was investigated. Morphologic studies of early growth-phase cultures and cells growing under temperature stress showed elongated cells typical of FtsZ inhibition/deficiency. Both ftsZ silencing and over-expression produced comparable growth effects in control cells, and altered the growth changes observed otherwise in the hok/sok+ cells. These changes were diminished in SOS-deficient strain containing mutant FtsZ. The involvement of FtsZ in the hok/sok-induced growth changes may be exploited as drug target in host bacteria, which often propagate antibiotic resistance elements.

A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells.

The major difference between viruses and plasmids is the mechanism of transferring their genomic information between host cells. Here, we describe the archaeal plasmid pR1SE from an Antarctic species of haloarchaea that transfers via a mechanism similar to a virus. pR1SE encodes proteins that are found in regularly shaped membrane vesicles, and the vesicles enclose the plasmid DNA. The released vesicles are capable of infecting a plasmid-free strain, which then gains the ability to produce plasmid-containing vesicles. pR1SE can integrate and replicate as part of the host genome, resolve out with fragments of host DNA incorporated or portions of the plasmid left behind, form vesicles and transfer to new hosts. The pR1SE mechanism of transfer of DNA could represent the predecessor of a strategy used by viruses to pass on their genomic DNA and fulfil roles in gene exchange, supporting a strong evolutionary connection between plasmids and viruses.

Bacteriophage P1vir-induced cell-to-cell plasmid transformation in Escherichia coli.

Bacteria undergo horizontal gene transfer via various mechanisms. We recently reported that cell-to-cell transfer of nonconjugative plasmids occurs between strains of Escherichia coli in co-cultures, and that a specific strain (CAG18439) causes frequent plasmid transfer involving a DNase-sensitive mechanism, which we termed “cell-to-cell transformation”. Here we found that CAG18439 is a type of P1 bacteriophage lysogen that continuously releases phages. We tested the ability of P1vir bacteriophage to induce horizontal plasmid transfer and demonstrated that such a horizontal plasmid transfer was caused by adding culture supernatants of P1vir-infected cells harboring plasmids to other plasmid-free cells. This plasmid transfer system also reproduced the major features of plasmid transfer involving CAG18439, suggesting that P1vir-induced plasmid transfer is equivalent or very similar to plasmid transfer involving CAG18439. We further revealed that approximately two-thirds of the P1vir-induced plasmid transfer was DNase-sensitive, but that complete abolition of plasmid transfer was observed when proteins were denatured or removed, despite the presence or absence of DNase. Therefore, we concluded that P1vir-induced plasmid transfer is largely due to the occurrence of cell-to-cell transformation, which involves the assistance of some proteinaceous factor, and partly due to the occurrence of plasmid transduction, which is mediated by phage virions. This is the first demonstration of the P1-phage-induced cell-to-cell transformation.

Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria.

Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein.

Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L−1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers.

Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50–90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity.

Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System.

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016.

CDI Systems Are Stably Maintained by a Cell-Contact Mediated Surveillance Mechanism.

Contact-dependent growth inhibition (CDI) systems are widespread amongst Gram-negative bacteria where they play important roles in inter-cellular competition and biofilm formation. CDI+ bacteria use cell-surface CdiA proteins to bind neighboring bacteria and deliver C-terminal toxin domains. CDI+ cells also express CdiI immunity proteins that specifically neutralize toxins delivered from adjacent siblings. Genomic analyses indicate that cdi loci are commonly found on plasmids and genomic islands, suggesting that these Type 5 secretion systems are spread through horizontal gene transfer. Here, we examine whether CDI toxin and immunity activities serve to stabilize mobile genetic elements using a minimal F plasmid that fails to partition properly during cell division. This F plasmid is lost from Escherichia coli populations within 50 cell generations, but is maintained in ~60% of the cells after 100 generations when the plasmid carries the cdi gene cluster from E. coli strain EC93. By contrast, the ccdAB "plasmid addiction" module normally found on F exerts only a modest stabilizing effect. cdi-dependent plasmid stabilization requires the BamA receptor for CdiA, suggesting that plasmid-free daughter cells are inhibited by siblings that retain the CDI+ plasmid. In support of this model, the CDI+ F plasmid is lost rapidly from cells that carry an additional cdiI immunity gene on a separate plasmid. These results indicate that plasmid stabilization occurs through elimination of non-immune cells arising in the population via plasmid loss. Thus, genetic stabilization reflects a strong selection for immunity to CDI. After long-term passage for more than 300 generations, CDI+ plasmids acquire mutations that increase copy number and result in 100% carriage in the population. Together, these results show that CDI stabilizes genetic elements through a toxin-mediated surveillance mechanism in which cells that lose the CDI system are detected and eliminated by their siblings.

Cloning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene in Anabaena sp. PCC7120

Human granulocyte-macrophage colony stimulating factor(hGM-CSF) is one of a family glycoprotein cytokines that have potent effects in stimulating proliferation,maturation and function of hematopoietic cells.The gene encoding hGM-CSF was PCR amplified from the plasmid pAGMT-8 and modified by addition SD sequence to promote prokaryotic expression.The resultant hGM-CSF was inserted into downstream of the strong promoter,PpsbA of expression vector pRL-439,then ligated with pDC-08 to produce the shuttle expression vector,pDC-GM.The resulting pDC-GM was transferred into the filamentous,heterocystour cyanobacterium,Anabaena PCC7120,by the tri-parental conjugation transfer method.When cells were cultivated without antibiotics for many generations,the transgenic cyanobacteria remained resistant to neomycin.PCR amplification of wild type and transgenic Anabaena cells with primers P_1 and P_2 revealed no band in wild type and plasmid free cells,whereas transgenic cells yielded a about(390 bp) band.This is the first report of expression hGM-CSF in cyanobacteria cells.

Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids.

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. 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.

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems.

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.

박테리아의 toxin-antitoxin system과 생명공학기술 응용

Toxin-antitoxin (TA) systems are ubiquitous genetic modules that are evolutionally conserved in bacteria and archaea. TA systems composed of an intracellular toxin and its antidote (antitoxin) are currently classified into five types. Commonly, activation of toxins under stress conditions inhibits diverse cellular processes and consequently induces cell death or reversible growth inhibition. These effects of toxins play various physiological roles in such as regulation of gene expression, growth control (stress response), programmed cell arrest, persister cells, programmed cell death, phage protection, stabilization of mobile genetic elements or postsegregational killing of plasmid-free cells. Accordingly, bacterial TA systems are commonly considered as stress-responsive genetic modules. However, molecule screening for activation of toxin in TA system is available as development of antimicrobial agents. In addition, cytotoxic effect induced by toxin is used as effective cloning method with antitoxic effect of antitoxin; consequently cells containing cloning vector inserted a target gene can survive and false-positive transformants are removed. Also, TA system is applicable to efficient single protein production in biotechnology industry because toxins that are site-specific ribonuclease inhibit protein synthesis except for target protein. Furthermore, some TA systems that induce apoptosis in eukaryotic cells such as cancer cells or virus-infected cells would have a wide range of applications in eukaryotes, and it will lead to new ways of treating human disease. In this review, we summarize the current knowledge on bacterial TA systems and their applications.

Curing a large endogenous plasmid by single substitution of a partitioning gene

To investigate whether plasmid-free cells of pathogenic Escherichia coli can be isolated by disrupting a single gene in an endogenous plasmid without further treatment, the effect of the disruption of partitioning genes on the inheritance of the endogenous plasmid pUTI89 of the uropathogenic E. coli strain UTI89 was studied. We found that mutation of parB, which encodes a type Ib partitioning protein, could cause loss of the endogenous plasmid at a ratio of about 1%. Clones derived from parB mutants, identified by antibiotic sensitivity, were all plasmid free. Plasmid instability caused by the parB mutation was found to correlate with a negative effect on host cell growth. Thus, in this pathogenic E. coli, an endogenous plasmid as large as 114kbp could be cured effectively by targeting a single type Ib partitioning gene followed by passaging, which may facilitate further investigations on the function of endogenous plasmids in their natural hosts.

The role of the hok/sok locus in bacterial response to stressful growth conditions

The hok/sok locus is renowned for its plasmid stabilization effect via post-segregational killing of plasmid-free daughter cells. However, the function(s) of the chromosome-encoded loci, which are more abundant in pathogenic strains of a broad range of enteric bacteria, are yet to be understood. Also, the frequent occurrence of this toxin/antitoxin addiction system in multi-drug resistance plasmids suggests additional roles. In this study, the effects of the hok/sok locus on the growth of bacteria in stressful growth-limiting conditions such as high temperature and antibiotic burden were investigated using hok/sok plasmids. The results showed that the hok/sok locus prolonged the lag phase of host cell cultures, thereby enabling the cells to adapt, respond to the stress and eventually thrive in these growth-limiting conditions by increasing the growth rate at exponential phase. The hok/sok locus also enhanced the survival and growth of cells in low cell density cultures irrespective of unfavourable growth conditions, and may complement existing or defective SOS mechanism. In addition to the plasmid stabilization function, these effects would enhance the ability of pathogenic bacteria to establish infections and propagate the antibiotic resistance elements carried on these plasmids, thereby contributing to the virulence of such bacteria.

Conditional Activation of Toxin-Antitoxin Systems: Postsegregational Killing and Beyond.

Toxin-antitoxin (TA) systems are small genetic modules formed by a stable toxin and an unstable antitoxin that are widely present in plasmids and in chromosomes of Bacteria and Archaea. Toxins can interfere with cell growth or viability, targeting a variety of key processes. Antitoxin inhibits expression of the toxin, interacts with it, and neutralizes its effect. In a plasmid context, toxins are kept silent by the continuous synthesis of the unstable antitoxins; in plasmid-free cells (segregants), toxins can be activated owing to the faster decay of the antitoxin, and this results in the elimination of these cells from the population (postsegregational killing [PSK]) and in an increase of plasmid-containing cells in a growing culture. Chromosomal TA systems can also be activated in particular circumstances, and the interference with cell growth and viability that ensues contributes in different ways to the physiology of the cell. In this article, we review the conditional activation of TAs in selected plasmidic and chromosomal TA pairs and the implications of this activation. On the whole, the analysis underscores TA interactions involved in PSK and points to the effective contribution of TA systems to the physiology of the cell.

COEXISTENCE OF EVOLVING BACTERIA STABILIZED BY A SHARED BLACK QUEEN FUNCTION

The Black Queen Hypothesis (BQH) was originally proposed to explain the dependence of some marine bacteria on helper organisms for protection from hydrogen peroxide (HOOH). The BQH predicts that selection for the evolutionary loss of leaky functions from individuals can produce commensal or mutualistic interactions. We demonstrated the leakiness of HOOH detoxification by complementing a HOOH-sensitive Escherichia coli mutant with a plasmid-encoded HOOH-detoxifying enzyme, KatG, and then evolving populations founded by this strain in two environments. When HOOH was absent, plasmid-carrying cells were outcompeted by plasmid-free segregants, reflecting the high cost of KatG expression. However, plasmid-carrying and plasmid-free cells coexisted for at least 1200 generations in three replicate populations evolved in the presence of HOOH, although their relative proportions fluctuated as beneficial mutations arose in one type or the other. Evolved plasmid-bearing cells reduced the cost of plasmid carriage even as they increased the rate of HOOH removal relative to the ancestor. Meanwhile, plasmid-free cells remained dependent on HOOH detoxification by the plasmid-bearing cells. These results demonstrate that partitioning of a Black Queen function can enable the stable coexistence of very similar organisms, even in this most restrictive case where the two types are competing for a single resource.

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcusneoformans

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1-A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5-fluoroorotic acid treatment of ORF4-expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid-free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1-A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.

New quantitative methods for measuring plasmid loss rates reveal unexpected stability

Plasmid loss rate measurements are standard in microbiology and key to understanding plasmid stabilization mechanisms. The conventional assays eliminate selection for plasmids at the beginning of the experiment and screen for the appearance of plasmid-free cells over long-term population growth. However, it has been long appreciated in plasmid biology that the growth rate differential between plasmid-free and plasmid-containing cells at some point overshadows the effect of primary loss events, such that the assays can greatly over-estimate inherent loss rates. The standard solutions to this problem are to either consider the very early phase of loss where the fraction of plasmid-free cells increases linearly, or to measure the growth rate difference either by following the population for longer time or by measuring growth rates separately. Here we mathematically show that in all these cases, seemingly small experimental errors in the growth rate estimates can overshadow the estimates of the loss rates. For many plasmids, loss rates may thus be much lower than previously thought, and for some plasmids, the estimated loss rate may have nothing to do with actual loss rates. We further modify two independent experimental methods to separate inherent losses from growth differences and apply them to the same plasmids. First we use a high-throughput microscopy-based approach to screen for plasmid-free cells at extremely short time scales – tens of minutes rather than tens of generations – and apply it to a par− version of mini-R1. Second we modify a counterselection-based plasmid loss assay inspired by the Luria–Delbrück fluctuation test that completely separates losses from growth, and apply it to various R1 and pSC101 derivatives. Concordant results from the two assays suggest that plasmids are lost at a lower frequency than previously believed. In fact, for par− mini-R1 the observed loss rate of about 10−3 per cell and generation seems to be so low as to be inconsistent with what we know about the R1 stabilization mechanisms, suggesting these well characterized plasmids may have some additional and so far unknown stabilization mechanisms, for example improving copy number control or partitioning at cell division.