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

Need we optimize the mating system when deal with every different genus？

Assessment of horizontal gene transfer in Lactic acid bacteria – A comparison of mating techniques with a view to optimising conjugation conditions
Niamh Toomey, Áine Monaghan, Séamus Fanning, Declan J. Bolton
Journal of Microbiological Methods, 2009, 77: 23–28

The most common laboratory techniques used to assess HGT in vitro include: plate, filter and broth mating protocols. While there is a general acceptance that higher transfer frequencies occur when using solid-phase mating mediums (since conjugation requires mating cells to be in close contact with each other), few studies have experimentally compared HGT techniques, with a view to standardizing these approaches, thereby providing meaningful direct comparisons.
In this study, plate, filter and broth mating techniques were assessed and compared by using a taxonomically diverse group, Lactic acid bacteria (LAB). LAB is a Gram-positive, catalase-negative group, which share the capacity to ferment sugars into lactic acid. Due to their aerotolerant anaerobic nature they are found in a variety of different environments. LAB also has the potential to act as resistance gene reservoirs with the ability to transfer these genes in a range of different environments; including transfer to pathogenic species. Genes conferring resistance to antimicrobials such as tetracycline, erythromycin, streptomycin, chloramphenicol, and vancomycin, have been found in LAB isolated from foods. These resistance genes were found to be located on transferable elements including plasmids and conjugative transposons. In present study, one L. lactis strain with the broad-host range plasmid pAMβ-1 [containing an erythromycin resistance marker, erm(B)], and two L. lactis strains with the conjugative transposon Tn916 [expressing a tetracycline resistance gene, tet(M)] were used as the donor strains, along with a marked (Strr, Rifr) L. lactis strain as recipient.
The plate mating technique used in this study is almost the same with that used in TopLab, except that following overnight incubation and scraping of the bacterial spots, additional medium was used to wash the plate for accurately calculating the transfer frequency. The filter mating technique is exactly the same with that we used. The broth mating method is just to add equal volume donor culture and recipient culture into one tube, and then following the overnight incubation, directly dilute the mixed culture and spread onto selective plates. Transconjugants were confirmed as the true transconjugants but not the reverted mutants by using antibiotic selection, E-tests to determine MICs, PCR assays to detect the corresponding marker genes, DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), and Southern blotting.
Based on the results of transfer frequency, the general trend is plate > filter > broth. In addition, in most cases, there is no significant difference between plate mating and filter mating. Effects of different pH, varying from 6.0 to 8.0, on the transfer rate were also detected. The pH of medium between 6.0 and 7.0 had no significant effect on transfer rate, while at pH 8.0 the conjugation was completely inhibited.
As the author said, this paper is the first study to examine the influence of mating protocols on both plasmid and transposon conjugal transfer between lactococcal strains. The results provide the detailed information about which system should be recommended in future LAB HGT studies.
However, some of the other research works are not in agreement with the results from this study. Both Lampkowska et al. (2008) and Langella et al. (1996) suggested that the filter facilitates a greater degree of donor-recipient contact than the plate method. Therefore, a question rises up. Need we optimize the mating system (including the mating techniques and environmental factors, such as temperature, pH) when we deal with every different genus, such as Pseudomonas, Agrobacterium, and Cupriavidus, which currently used in our lab? If it does, then a great amount of work we should do. Do you think it is necessary? Feel free to take part in the discussion. It is a Blog, and there should be some discussion here.



Additional reference:

Lampkowska, J., Feld, L., Monaghan, A., Toomey, N., Schjørring, S., Jacobsen, B., van der Voet, H., Andersen, S.R., Bolton, D., Aarts, H., Krogfelt, K.A., Wilcks, A., Bardowski, J., 2008. A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species. Int. J. Food Microbiol. 127, 172–175.

Langella, P., Zagorec, M., Ehrlich, S.D., Morel-deville, F., 1996. Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake. FEMS Microbiol. Lett. 139, 51–56.



Hui Li, PhD
University of Idaho

The defective prophage pool of Escherichia coli O157: Prophage-prophage interactions potentiate horizontal transfer of virulence determinants

Asadulghani M. and Ogura Y. et al, (2009) PLOS Pathogens 5: 1-15.

Bacteriophage is one of the major genetic elements promoting horizontal gene transfer between bacteria. Enterohemorrhagic Escherichia coli O157:H7 (Sakai isolate) contains eighteen prophages in its genome (Ohnishi et al. 1999). Two of the eighteen prophages carry Shiga toxin gene clusters; stx1AB and stx2AB; these gene products kill eukaryotic cells by inhibiting protein synthesis. Interestingly, all prophages in O157 have mutations in genes that encode basic phage functions such as site-specific recombination, replication, cell lysis, or structural proteins constituting the head and tail. Evidence obtained by genome analysis suggests that each prophage is defective. Thus, one might think this strain is not very problematic. But, it may be not true.


To assess the role of defective prophages in horizontal gene transfer of virulence determinants, the authors analyzed each basic function of the prophages in O157: excision from chromosome, phage DNA amplification in response to DNA damage, ability to be released as phage particles, and infection of new recipient cells.

Among eighteen prophages, eight prophages including the stx gene-containing phages were found to be released as phage particles, and among them four were able to infect both or either of the two E. coli recipient strains tested. These results strongly suggest that prophages in O157 are complementing defective functions with each other to make practically transferable phages.


Importantly, the phage particle produced often contains a chimeric DNA fragment made of two different prophages' fragments, indicating the recombination of prophages in host cells. Although the newly generated phage DNA were not extensively sequenced in this study, it is possible that some phage fragments reconstituted intact, functional phage through recombination.


This paper nicely explains the way bacteriophages live and propagate in a host. I think this paper is a good example of a post-genomic sequence study. In this era, we can understand how mobile genetic elements are acting in a population by combining sequence analysis and standard genetic experiments. I suspect that plasmids and transposons are also repeating cycles of inactivation and activation as shown for these phages. They will never be extinguished as long as similar mobile genetic elements exist in the world.


Additional reference:
Ohnishi M. and Tanaka C. et al., (1991) DNA Res. 6: 361-368


Posted by H. Yano, Univerisity of Idaho

Plasmid Conjugation in an Activated Sludge Microbial Community

By Ruoting Pei and Claudia K. Gunsch

ENVIRONMENTAL ENGINEERING SCIENCE,

Vol 26, No 4, 2009, p. 825

This is jet another paper describing plasmid transfer and their role in so called bioaugmentation in natural kind of environments.

Bioaugmentation involves the addition of exogenous micro-organisms that have the ability of degrading the compound of interest. Using this process, new biodegradation pathways can be added, which inherently improve the metabolic conversion of the contaminants. The main problem of this method is that the laboratory strains used to introduce new genetic traits usually cannot grow in natural environmental conditions. This means that bioaugmentation can fail due to the poor establishment and=or survival of new strains under field environmental conditions. The second process used for enhancing natural biodegradation capabilities is biostimulation which consists of adding nutrients (e.g., carbon, nitrogen, electron acceptor, etc.) to promote the growth of indigenous microorganisms. This method is preferred over bioaugmentation because no additional bacterial strains are introduced but it requires the presence of indigenous microorganisms, which are capable of breaking down the contaminant of interest. To overcome the limitations associated with biostimulation and bioaugmentation, it might be possible to combine these two strategies into a technique called genetic bioaugmentation. This technique would consist of adding bacteria with the needed genes and inducing their horizontal gene transfer (HGT) to indigenous bacterial species utilizing natural prokaryotic adaptation mechanisms.

In this paper authors described pWWO plasmid transfer from Pseudomonas putida strain to bacterial population present in activated sludge. The pWWO plasmid is well known for its toluene degradation capability. The plasmid DNA was tagged with transposone containing gfp gene driven by lac promoter. In the donor strain which overproduce LacI repressor gfp is not expressed. In other bacterial hosts gfp is expressed and gives bright green fluorescence which can be measured. Using flow cytometry authors showed that plasmid was actually transferred into bacterial community of activated sludge with number of bacteria expressing GFP protein up to 6%. They showed that the highest number of transconjugants is reached at the third day of experiment and it depends on the donor to recipient ratio with the best results at the 1:20 ratio. Authors tried to connect the plasmid transfer with the actual capability of activated sludge to toluene degradation. This experiment did not really work as the level of toluene degradation remained unchanged during the time of the experiment.

The paper stays in a main stream of microbial ecology and engineering facing the problem of global pollutions and waists utilization. It is very important to use new methods for detecting plasmid transfer in natural environment. Authors were facing the same problem as in previously published papers that there are some limitations in using fluorescent proteins as a plasmid infection marker. The true plasmid bearing cells are not known until gfp gene is expressed and protein is properly matured to give strong fluorescence, which can be detected by flow cytometry. Authors cannot also link the presence of TOL phenotype (toluene degradation) with the number of plasmid bearing cells as the sludge microbial community shows already high toluene degradation activity.

Perhaps, it is not a very “big” paper, but it is a small step toward understanding how the plasmid transfer occurs in natural environment and how can we use this to save our polluted world.

Jaroslaw Krol, PhD

UofI

The SOS Response Controls Integron Recombination

Émilie Guerin, Guillaume Cambray, Neus Sanchez-Alberola, Susana Campoy, Ivan Erill, Sandra Da Re, Bruno Gonzalez-Zorn, Jordi Barbé, Marie-Cécile Ploy, and Didier Mazel

Science, May 2009 p 1034, Vol. 324, No. 5930.

An ever-growing concern in the medical world is the rise of antibiotic-resistant pathogens. One source of comfort that we have had in this fight against the spread of resistance is that the expression of resistance genes confers a cost to bacteria. In the absence selection for antibiotics it is thought that this cost will give a selective advantage to non-resistant bacteria, and so bacterial populations will loose resistance over time in the absence of antibiotics. The authors of this paper suggest that this is unfortunately not always the case.

In the words of Jurassic Park, “Nature will find a way.” For bacteria, one of the weapons in their arsenal against antibiotics is the SOS response. Under normal conditions the protein LexA binds to the operator region controlling SOS genes to repress their expression. In the event of DNA damage LexA is cleaved from this region and a series of DNA repair mechanisms are activated, some of which are low- fidelity and therefore lead to an increase of mutations, some of which may be beneficial in resisting further DNA damage.

In terms of antibiotic resistance, this paper explains that LexA repression and SOS expression also influences recombination of genes cassettes (which often code for antibiotic resistance. During SOS expression cassette recombination is induced. This can result in either silencing or reactivation of cassettes. In other words, a cassette carrying resistance genes can be reactivated through recombination under times of stress and then revert to a “dormant” form that provides no cost to the bacterium once the SOS response is again repressed.


Additional Reading:
Erill, S. Campoy, J. Barbe, FEMS Microbiol. Rev. 31, 637 (2007).
C. M. Collis, R. M. Hall, Antimicrob. Agents Chemother. 39, 155 (1995).
A. Aertsen, C. W. Michiels, Trends Microbiol. 14, 421 (2006).
D. I. Andersson, Curr. Opin. Microbiol. 9, 461 (2006).


Julie Hughes,
Graduate Student, University of Idaho