Sebastien Bonot and Christophe Merlin
Applied and Environmental Microbiology Vol. 76 No. 1 2010
There have been many discoveries about the process of horizontal gene transfer and our understanding of this subject has increased greatly in recent years. However, the work that can be done on this process is hindered by limitations inherent in laboratory settings. The results of current experiments are difficult to apply to real-world situations with diverse microbial communities. For example, it is believed that we are only able to cultivate approximately 1% of environmental bacteria under laboratory conditions. Also, genetic information transferred under laboratory conditions may show narrow host expression. Quantifications using culture-based techniques may lead to an underrepresentation of the extent of gene transfer that has occurred, especially in complex environments. Recent work involving green fluorescent protein allows us to see a more realistic representation of the gene transfer that would occur in these complex environments. However, the authors suggest that these studies are still not completely representative of natural environments because the elements under study still require genetic manipulation. PCR and other molecular techniques can get around these problems, but are not typically used due to the fact that so many DNA markers are shared among different genomes. In this paper the authors show that with carefully designed, specific primers they were able to use quantitative PCR (qPCR) to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. E. coli DH5α was chosen to be the donor strain because it had poor chances of survival since the sediment microcosms under study were not its natural habitat. Also, the high numbers of genetic alterations that this lab strain has undergone were likely to further reduce the donor’s chances of survival.
To follow the fate of plasmid pB10 with qPCR the researchers needed to develop primers specific to the plasmid, as well as to the donor bacteria, E. coli DH5α. The researchers were unable to develop traditional primers specific to pB10 due to the fact that the plasmid has many similarities to other genetic elements. The researchers came up with a very interesting solution to this problem. They used the fact that bacterial genomes have a unique structure based on a combination of DNA blocks instead of just specific DNA sequences. The researchers developed unique primers for pB10 that prime on both sides of a junction of these building blocks. They used the same technique to design primers for DH5α. qPCR tests were performed on total environmental DNA to determine whether or not these primers were truly specific. Amplification was only achieved when DNA was present from pB10 or DH5α.
Tests were run to determine the ability of qPCR to quantify DNA from pB10 and DH5α when in complex environments. Samples were inoculated with known amounts of DH5α/pB10, followed by immediate total DNA extraction and qPCR quantification. 20% of the expected pB10 DNA was recovered, and 0.25% of DH5α DNA was recovered. The researchers postulate that this discrepancy occurred because plasmid DNA is much easier to recover than chromosomal DNA. They also determined that these results mean they must analyze qPCR results based on appearance/disappearance of DNA instead of absolute quantities.
The researchers then performed an experiment to monitor the fate of pB10 in sediment microcosms. The microcosms used were blended river and sediment samples. These were inoculated with pB10/DH5α. Microcosms were maintained for 5 days and total community DNA was sampled at intervals. The researchers found that pB10 concentrations remained stable, while DH5α DNA was lost completely after 48 hours. This suggests that pB10 invaded the microcosm relatively early, before DH5α was lost. Controls that were inoculated with naked pB10 DNA showed a rapid loss of plasmid DNA that disappeared completely after 48 hours. This shows that the plasmid could not have been in the environment extracellularly during the experiment.
This experiment is noteworthy because it monitors the dissemination of a broad-host-range plasmid in a complex environment using molecular tools. I found the use of DNA block primers over conventional sequence primers quite interesting. Perhaps this will lead to new methods of studying horizontal gene transfer in complex environments. The use of qPCR to do this would allow researchers to study horizontal gene transfer in these environments with minimal disturbances to the microbial communities. The researchers plan to expand on their current work by including factors such as spatial structure in future analyses.
Ryan Simmons
University of Idaho
Friday, February 19, 2010
Tuesday, February 9, 2010
Acquisition of prokaryotic genes by fungal genomes
Marina Marcet-Houben and Toni Gabaldon
Trends in Genetics Vol. 26 No. 1 2010
Horizontal gene transfer between bacteria is widely studied for its numerous consequences including increased antibiotic resistance, virulence and transfer of metabolic pathways. What is less often considered is horizontal gene transfer from a prokaryotic donor to a eukaryotic recipient. Although this is known to happen, its mechanisms and effects are poorly understood. This study searches for genes of bacterial origin in the fungal kingdom to attempt to gain a broader understanding of the frequency of gene transfer from prokaryotes to fungus and what its evolutionary implications might be.
This study was conducted using whole genomes of over 60 different fungi and over 600 genomes from prokaryotic and other eukaryotic organisms. Using a conservative detection method they found 713 genes over 53 genomes that were acquired from prokaryotic sources. This is an indication of the frequency of horizontal gene transfer to eukaryotes, but also represents a novel way to search for horizontal gene transfer events using whole genomes. However, the number of transfer events is difficult to estimate due to gene loss and multiple genes being transferred in a single event.
The distribution of horizontal gene transfer events over the different fungal clades is not even and suggests factors that could aid or hinder transfer. Identification of traits in eukaryotic species that make them good candidates for horizontal gene transfer could have far reaching implications. Several of the genes that were observed to transfer were analyzed and their possible evolutionary advantages discussed. The first to be discussed was the arsenic detoxification pathway. It appears that the specific types of yeast mentioned have the machinery to reduce arsenate to arsenite but a bacterial reductase successfully transferred and replaced the standard yeast reductase. In another example, to convert between optical isomers of amino acids a racemase is necessary. Several different types were found to be transferred into two different members of the yeast family and a rotifer from a bacterial species. This could lead to the ability to use new sources of amino acids. Bacterial catalases were also found to be transferred to pathogenic fungal species. These catalases help protect pathogens from host reactive oxygen defense mechanisms. Finally, the transfer of a functional bacterial metabolic pathway was found in Aspergillus species. What is remarkable about this transfer is that the three genes that make up this pathway appear to have moved as two units, with two of the fused into a single gene, instead of three separate genes as they are found in the donor.
This study has a well thought out approach to searching for horizontal gene transfer events between prokaryotes and eukaryotes that could allow insight into the evolutionary history of many different species with unique abilities. It will be interesting to see if this type of horizontal gene transfer has played a larger role in the evolution of eukaryotes than previously thought.
Brian Lohman
University of Idaho
Saturday, January 23, 2010
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
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
Tuesday, January 12, 2010
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
Wednesday, October 7, 2009
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
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
Monday, October 5, 2009
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
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
Friday, September 25, 2009
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
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
Subscribe to:
Posts (Atom)