Mobilization and prevalence of a fusobacterial plasmid

Mobilization and prevalence of a fusobacterial plasmid.
Brianna M. Claypool, Sean C. Yoder, Diane M. Citron, Sydney M. Finegold, Ellie J.C. Goldstein and Susan Kinder Haake
Plasmid 2010 63:11-19

Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections.
Studies indicate considerable phenotypic variability between F. nucleatum strains, and biochemical analyses have suggested that F. nucleatum represents a “species complex”. Comparative genomic analyses revealed that 25% of the genes encoded by F. nucleatum ATCC 10953 ssp. polymorphum are not found in either of the sequenced genomes of F. nucleatum ssp. nucleatum and vincentii). In addition, 21% of these unique ORFs mapped to clusters of five or more genes, suggesting that they may have been introduced into the genome by horizontal gene transfer. Several plasmids were found in Fusobacterium but they are too small to be self transmissible.
In this paper authors defined the minimal “mobilon” of plasmid pFN1 that included the relaxase, and DNA directly upstream containing ORF4 and ORF7 with the oriT region located upstream of ORF4. The pFN1 mobilon is related by sequence to the mobilons of the staphylococcal plasmids pC221 and pC223. Authors screened 94 clinical and 4 laboratory isolates of F. nucleatum for plasmids and plasmid encoded relaxase gene and they found that 11.5% of isolates.
As the main discovery authors demonstrated for the first time that the fusobacterial plasmid pFN1 encodes genes enabling efficient mobilization between strains of E. coli by the broad-host range plasmid RP4. I found this experiment really interesting. It is true that IncP plasmid RP4 can mobilize plasmid pFN1 between E. coli strains but if we look closer we can find that plasmid pFN1 cannot replicate in E. coli as oriP15A was used for stable replication. On the other hand there is no evidence that pRP4 can transfer to and/or replicate in F. nucleatum. This can raise a question, how is it possible for pRP4 to mobilize the pFN1 plasmid in vivo. Unfortunately, authors did not discuss that point. On the other hand they realize that the main aim to really prove the role of Fusobacterium plasmids in horizontal gene transfer is to show that plasmid transfer occurs in vivo and find what can support the missing conjugation functions.
Our dual reporter system for detection plasmid transfer in situ in anaerobic conditions can help in this research.

Jarek Krol
UofI

Into the Woods

The fundamental units, processes and patterns of evolution, and the Tree of Life conundrum

Eugene V Koonin and Yuri I.Wolf

Biology Direct (2009), 4:33

Human communication and thought is largely shaped by metaphors. Metaphors allow man to think about concepts and facts in new ways and to make connections that otherwise might have been left undiscovered. However, if one becomes too committed to a particular metaphor there is the danger of the opposite-of stifling creativity and having a distorted view of individual facts or even the world at large.

"The affinities of all the beings of the same class have sometimes been represented by a great tree. I believe this simile largely speaks the truth. The green and budding twigs may represent existing species; and those produced during each former year may represent the long succession of extinct species. .... The limbs divided into great branches, and these into lesser and lesser branches, were themselves once, when the tree was small, budding twigs; and this connexion of the former and present buds by ramifying branches may well represent the classification of all extinct and living species in groups subordinate to groups."

The authors of this article begin with this quote from Charles Darwin(1) in order to assert that the very roots of evolutionary theory are grounded in the metaphor of a “Tree of Life.” In this metaphor all living organisms are the budding tips of living branches in the Tree of Life (TOL), whose trunk was the original organism from which all life derived. While this metaphor has served scientists well for centuries, our increased understanding of horizontal gene transfer has led to a “crisis of the TOL.”

The authors argue that individual viruses, plasmids, transposons, individual genes, and the like, rather than organisms or species, should be considered as the true fundamental units of evolution (FUEs). The evolutionary history of each individual FUE is still accurately represented by a tree in that an ancestral gene can be altered such that a new version “branches off,” with this process continuing until a full tree is formed. An organism, therefore, is made up of many such FUE trees, and so is more properly a “forest of life.”

The more we learn about the importance of horizontal gene transfer among organisms, species, or even kingdoms, the more clear it becomes that the metaphor of a Tree of Life ignores much of the complexity of true evolutionary histories, especially among prokaryotes. We therefore might benefit from reevaluating our metaphor and perhaps, as the authors suggest, acknowledge the true complexity of the Forest of Life.

References\Further Reading:

1. Darwin C: On the Origin of Species. 1st edition. London: Murray; 1859.

2. Hilario E, Gogarten JP: Horizontal transfer of ATPase genes--the tree of life becomes a net of life. Biosystems31(2-3):111-119. 1993,

3. Doolittle WF: Uprooting the tree of life. Sci Am 2000, 282(2):90-95

4. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, et al.: Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 1999, 399(6734):323-329.

Julie M. Hughes

University of Idaho

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry.

W. Florian Fricke, Patrick F. McDermott, Mark K. Mammel, Shaohua Zhao, Timothy J. Johnson, David A. Rasko, Paula J. Fedorka-Cray, Adriana Pedroso, Jean M. Whichard,
J. Eugene LeClerc, David G. White, Thomas A. Cebula, and Jacques Ravel


Salmonella enterica is a common cause of food –borne gastroenteritis. This combined with the rise of multidrug resistant S. enterica isolates is a grave medical concern. The S enterica subsp. enterica serovar Kentucky is the most common serotype found in chickens [1,2]. Moreover this serotype is often found to be resistant to antibiotics such as tetracycline and streptomycin [2]. The goal of the study was to find clues to the development of multi-drug resistance in S. Kentucky. The authors analyzed the complete sequences of the 3 large plasmids (pCVM29188_146, pCVM29188_101, pCVM29188_46) that they isolated from S. Kentucky CVM29188. Only the two large plasmids (pCVM29188_146 and pCVM29188_101) were found to carry antibiotic resistance genes. Thus, genes coding for resistance to aminoglycosides (strAB) and tetracyclins (tetRA) were found on pCVM29188_146 and those coding for resistance to cephalosporins (bla CMY-2) were found on pCVM29188_101. Both resistance plasmids (pCVM29188_101 and pCVM29188_146) in this study were found to have intact transfer regions, while the smaller plasmid pCVM29188_46 (46kb) did not have any transfer genes. Sequence similarity of the replication and transfer genes to other plasmids suggest that plasmid pCVM29188_101 may belong to the IncI1 group while plasmid pCVM29188_46 may belong to the IncFII group. The backbone of plasmid pCVM29188_146 is very similar to two plasmids isolated from avian pathogenic E. coli strains and also have the same virulence factors. The plasmid pCVM29188_46 has little similarity to other plasmids and has a lot of hypothetical proteins. They next conducted mating experiments with plasmids pCVM29188_146 and pCVM29188_101 and showed their transfer to two strains of Salmonella and a strain of E. coli. Next they wanted to test the abundance of the virulence genes on other isolates of S. Kentucky from meat, clinical and agricultural sources. So they screened 287 S. Kentucky isolates for the presence of virulence genes by PCR with primers specific for the 5 loci of pCVM29188_146 that were responsible for encoding virulence factors. They found that 64% of all S. Kentucky strains tested positive for the presence of at least one locus associated with virulence. The association was even stronger among the S. Kentucky strains that were isolated from chicken. This however was not the case for the 6 other Salmonella serovars that were isolated from chicken Moreover, all S. Kentucky strains that had at least one virulence locus of Pcvm29188_146 also had resistance to tetracycline and a subgroup of these had resistance to streptomycin. This suggests that a strong association may exist between S. Kentucky and plasmids like pCVM29188_146 that encode both virulence factors and resistance to antibiotics such as tetracycline and streptomycin. One explanation that the authors offer for this association is that pathogenic E. coli strains bearing virulence plasmids may have encountered resistance plasmids, leading to integration of the resistance genes into the virulence plasmid. This new plasmid could have transferred into a Salmonella strain such as the S. Kentucky commonly found in chickens. They do acknowledge that this does not explain why other Salmonella strains isolated from chicken do not have this plasmid type. The second explanation is that virulence plasmids may help S. Kentucky in coping with stress or other enterobacteria and hence, the association. This again does not explain why the association is only seen in S. Kentucky isolated from chicken.
This is an interesting study of plasmids from a strain of bacterium that has medical relevance to us. It is indeed surprising that there is such a clear association of the virulence and antibiotic resistance encoding plasmid with the S. Kentucky strain isolated from chicken. Their explanations for the association seem a little weak.

References:

1. FDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS). Retail meat annual report, 2006. FDA, Bethesda MD. http://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM073302.pdf
2. USDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric
Bacteria (NARMS). Veterinary isolates final report, slaughter isolates,
2006. USDA, Washington, DC. http://www.ars.usda.gov/sp2UserFiles/Place/66120508/NARMS/narms_2006/NARMS2006.pdf.

Diya Sen
University of Idaho

Mobile Microenvironments

Horizontal Transfer of the Tetracycline Resistance Gene tetM Mediated by pCF10 Among Enteroccus faecalis in the House Fly Alimentary Canal
Mastura Akhtar, Helmut Hirt, Ludek Zurek
Environmental Microbiology

Vectors have largely aided the spread of microorganisms. Vectors move bacteria from one place to another as they themselves go about their life cycle. The movements of a house fly would be a prime example of this type of vector. However, a role of the vector not considered as often is its role as a habitat for a bacterium itself. During transport, or as a permanent environment, bacteria encounter a unique combination of other bacteria and nutrients that only the vector could assemble. This provides for a microenvironment that can play a key role in the evolution of and dissemination of traits beneficial to bacterial species. While inside the fly these traits can be transferred horizontally through conjugation, transduction and transformation. This study considers how plasmid mediated horizontal gene transfer in the gut of a house fly can mediate tetracycline resistance to transfer between bacterial species.
Two strains of enterocci, bacteria normally residing in the gastrointestinal tract, were selected for donor and recipient. The donor contained the tetM gene to identify transformants using selective media. Flies were separated into two groups, one that received the donor first, via infected food supply, and the other received the recipient strain first. After twelve hours flies were given food source containing the opposite strain for one hour. Each group was then subdivided so that while flies were checked for the presence of donor, recipient and transformants over the next five days and half would have their eating appendage sterilized and half would not.
Results showed that regardless of whether the donor or recipient was introduced first, both groups established concentrations of donor and recipient cells that were similar. The was also no statistical difference in rate of gene transfer between the two groups. Concentration of donor and recipient cells in the digestive tract was similar to that of the surface sterilized, suggesting that observed donor, recipient and transformants were localized to the gut.
Transformants began to be detected 24 hours after both stains were combined. Their presence was screened for using selective media. Groups of flies were sterilized at the surface to eliminate the possibility of surface contamination and transformation outside the vector. Portions of the food supplied to the flies were periodically screened for transformants with very little occurrence, suggesting this did not play a key role. However, it is possible that transfer is taking place on the eating appendage. The conditions in which intestinal gene transfer are best suited are not well understood. The final possible explanation could be that transformants could be the product of high plasmid transfer rate and subsequent rapid clonal expansion of transformants.
Horizontal transfer in a vector could lead to the spread of various genes and provide a unique microenvironment for evolution. The house fly’s unique combination of contact with decaying organic matter and food provides ample opportunity for transfer of traits between bacteria evolved to live in harsh environments to those that are common in food. This potential introduces a need to better understand horizontal gene transfer and evolution in microenvironments that can directly affect humans.

Brian Lohman
University of Idaho

Interkingdom Horizontal Gene Transfer—a hot topic in recent years

Ancient Horizontal Gene Transfer from Bacteria Enhances Biosynthetic Capabilities of Fungi
Schmitt I, Lumbsch HT (2009) PLoS ONE 4(2): e4437. doi:10.1371/journal.pone.0004437


Until recently, the studies of gene transfer have been mostly focused on prokaryotes, and the process of gene transfer is assumed to be of limited significance to eukaryotes. The availability of diverse eukaryotic genome sequence data is dramatically changing our views on the important role gene transfer can play in eukaryotic evolution. The rapid increase in fungal sequence data has promoted this kingdom to the forefront of comparative genomics. As a result, interkingdom HGT became a hot topic in recent years. Whereas there is very few documented evidence for interkingdom HGT, but they did happen. Here, we will present an ancient interkingdom HGT event between bacteria and fungi.

The targeted gene discussed here is the polyketide synthase (PKSs) genes, which involved in antibiotic and mycotoxin production. Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms.

Bacteria and fungi commonly harbor a group of PKSs that consists of a single protein complex carrying all catalytic sites (typeI PKS). In this paper, the authors are focusing on a clade of fungal type I PKSs gene which is closely related to bacterial PKSs. Since 6-methylsalicylic acid synthase (6-MSAS) was the first PKS in this group to be characterized, this clade is also termed as‘‘6-MSAS-type PKS’’. The lichenized fungi, which are characterized by a sophisticated vegetative morphology and a rich polyketide metabolism, were selected as the research materials in this study. The total genomic DNA of the lichenized fungi, which collected from 12 different countries, were extracted, and then the KS domain of fungal 6MSAS-type PKS genes were amplified by a degenerate primer pair, LC3 and LC5c. The amplified fragments were cloned and sequenced, and then all sequences were subjected to BLAST searches. The alignment was analyzed in a Bayesian phylogenetic framework using MrBayes 3.1. The tree resulting from this analysis was used to determine the PKS clades most closely related to the fungal 6-MSAS group. To evaluate potential problems with outgroup selection, three alignments including different outgroups were compared.

As a result, 24 6-MSA synthase sequence tags from lichen-forming fungi were generated. The results from comparative phylogenetics support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. In the Discussion, the authors inferred that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase. Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi.

This study revealed the phylogenetic origin of the enigmatic fungal 6-MSAS-type PKS biosynthetic gene using comparative analysis. The results provide statistical support to the hypothesis that this PKS was transferred from an actinobacterial source into ascomycete fungi during an ancient HGT event. They also report the finding of 6-MSAS-type PKS genes in a variety of lichen-forming fungi, and speculate about the possible role of lichen symbionts in the evolution of this gene. Overall, this paper added solid evidence to the fact of interkingdom HGT.


Hui Li Ph.D University of Idaho

Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture.

Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture.

S. Pignato, M. A. Coniglio, G. Faro, F. X. Weill and G. Giammanco
Journal of Water and Health Vol 07 No 2 pp 251–258

Antibiotic resistant bacteria strains are permanent threat to human populations. Genes encoding antibiotic resistance are commonly located on mobile genetic elements like bacterial plasmids. Horizontal gene transfer (HGT) occurs in the environmental condition. The main mechanism of HGT seems to be bacterial conjugation. This process requires direct contact between plasmid bearing, donor strain and plasmid free recipients. The frequency of conjugation depends on a number of different factors. One of them is the cell density. As we could imagine possibility of meeting of two bacterial cells living in 1ml of water is much lower than if there are millions of different cells occupying the same volume. Density of bacterial populations in environmental samples varies markedly depending on sampling sites. Usually is not very high ~106 cfu/ml. One of the places where bacterial population reaches high densities are wastewater treatment plants. So study of spread antibiotic resistance encoding bacterial plasmids in the waste water is very important.
In presented paper authors pointed out that the guidelines for cleaning water used for irrigation requires treatments to remove pathogens that can cause enteric infections for crop consumers, producers and handlers. According to the microbiological guidelines for safe use of wastewater in agriculture developed by the World Health Organization(WHO) less than 0.1 intestinal nematode eggs must be detected in 1 litre, while up to 1,000 faecal coliform bacteria per 100 ml can be tolerated for unrestricted irrigation. In the United States, much stricter wastewater quality standards for irrigation are recommended by the Environmental Protection Agency but, lacking federal standards for the quality of reclaimed water, individual states have developed guidelines mainly based on the daily monitoring of faecal coliform bacteria on a single, 100-ml sample, assuming a predictive relationship between indicator microorganisms and pathogen presence.
Although wastewater treatments proved to be effective in eliminating Salmonella spp. and in reaching WHO microbiological standards for safe use of wastewater in agriculture, they were ineffective in reducing significantly the frequency of plasmid-mediated multiple antibiotic resistance in surviving E. coli.
It was shown that 22.71%, 19.41%, 16.84% and 14.28% out of 273 isolates were resistant respectively to ampicillin, tetracycline, sulfamethoxazole, and streptomycin. Some other antibiotic resistant strains were detected at low frequency (trimetoprim – 9.15%; nalidixic acid – 8%; chloramphenicol – 5.12% and kanamycin – 2.93%). Also multiple antibiotic resistance was present in 24.17% of the isolates. Antibiotic resistance was detected to be transferred by conjugation from 54% resistant strains. Three different plasmids with the sizes of 125kb, 54kb and 60kb were isolated from those strains. Also some other mobile elements like class 1 integrons were detected in resistant strains.

Since multiple antibiotic-resistant bacteria carrying integrons and conjugative R plasmids can constitute a reservoir of antibiotic-resistance genes in wastewater reclaimed for irrigation, risks for public health should be considered. Bacterial strains carrying R plasmids and integrons could contaminate crops irrigated with reclaimed wastewater and transfer their resistances to the consumers’ intestinal bacteria. So we should remember to wash every vegetables and fruits before we will eat them…

Jarek Krol
UofI

Competition favors reduced cost of plasmids to host bacteria

Rembrandt J. F. Haft, John E. Mittler, and Beth Traxler

The ISME Journal (2009) 3, 761-769

When it comes to their relationship with their hosts it can sometimes be difficult to define what plasmids are. They often encode beneficial traits that can be useful, or even vitally necessary, for their host bacteria. A bacterium that finds itself in the gut of a patient taking antibiotics, for instance, may require plasmid-encoded resistance in order to survive. However, despite the potential usefulness of a given plasmid, plasmid carriage also comes with certain costs. In certain environments the same plasmid that used to be essential for survival can become a burden to its host due to the energy required of the host for plasmid maintenance and upkeep. In such circumstances plasmids can be viewed as parasitic; they need the host to survive but only confer costs, not benefits, to the host. Many plasmids have therefore developed clever ways with which to ensure their survival in bacterial populations, even in the absence of external selective pressures for plasmid maintenance (e.g. the presence of antibiotics).

It’s easy to imagine that in the absence of such selective pressures plasmid-free bacteria would out-compete plasmid-bearing cells. Through vertical inheritance alone this would ensure the eventual loss of plasmids from a mixed population of plasmid-bearing and free cells. One tool that most plasmids have to combat this loss is the ability to pass copies of themselves to neighboring cells via the horizontal gene transfer mechanism of conjugation. Conjugation allows plasmids to infect new hosts such that even in the absence of selection plasmids can survive in a population, even as they reduce the fitness of their hosts. Yet many plasmids have developed systems that inhibit their own horizontal transfer. The authors of this paper used a combination of mathematical models and laboratory experiments to determine when and why a plasmid might benefit in repressing, rather than promoting, their own conjugation.

What the authors predicted in their models (and confirmed in their experiments) is that by reducing transfer frequency plasmids can also reduce the cost that they’re imposing on their hosts. While plasmids that don’t repress conjugation will spread through a bacterial population more quickly on their own than plasmids with functioning repression systems, when both types of plasmids are present the latter will eventually take become dominant. This is because in limiting their horizontal transfer they give the competitive edge to their hosts, which will be more fit and grow faster than the bacteria harboring the plasmids that transfer more frequently, and consequently these transfer-limited plasmids are spread mainly through vertical transfer.

This paper not only elucidates the mystery of the persistence of transfer repression systems in plasmids, but also has broader applications to parasitic strategies in general. The authors point out that for many parasites being slightly less virulent gives some parasites a competitive advantage over those that kill their host before they are able to spread to a new one. The mathematical model that they developed could therefore be applied to a much large scope of systems, of which plasmid transfer is just one.

Additional Reading:

Bahl MI, Hansen LH, Sorensen SJ. (2007). Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 266: 250-6.

De Gelder L, Ponciano JM, Joyce P, Top EM. (2007). Stability of a promiscuous plasmid in

different hosts: no guarantee for a long-term relationship. Microbiology 153: 452-63.

Dionisio F. (2005). Plasmids survive despite their cost and male-specific-phages due to

heterogeneity of bacterial populations. Evolution Ecol Res 7: 1-19.

Freter R, Freter RR, Brickner H. (1983). Experimental and mathematical models of

Escherichia coli plasmid transfer in vitro and in vivo. Infect Immun 39: 60-84.

Kerr B, Neuhauser C, Bohannan BJ, Dean AM. (2006). Local migration promotes competitive

restraint in a host-pathogen 'tragedy of the commons'. Nature 442: 75-8.

Kover PX, Clay K. (1998). Trade-off between virulence and vertical transmission and the

maintenance of a virulent plant pathogen. Am Nat 152: 165-175.

Turner PE, Cooper VS, Lenski RE. (1998). Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. Evolution 52: 315-329.

Julie Hughes

University of Idaho