Slater, Frances R.; Bruce, Kenneth D.; Ellis, Richard J.; Lilley, Andrew K.; Turner, Sarah L.
2008
Applied & Environmental Microbiology-74 (10). 3189-3197. doi:10.1128/AEM.02383-07
Bacteria live in natural environments that have a spatial structure where their movement is restricted by the surrounding soil or water. Such a spatial structure may create physicochemical gradients leading to heterogeneous patches. This heterogeneity is very important to the biodiversity of microorganisms as studied by Rainey and Travisano (1998). The present study by Slater et al aims to compare the effects of uniform and heterogeneous mercuric chloride HgCl(2) on a model community of plasmid-carrying and plasmid-free pseudomonads. The authors describe a novel experimental system for quantification of the different spatially variable selection pressures that are present in natural environments.
Plasmids may carry a variety of genes for antibiotic and heavy metal resistance that benefit their hosts. These genes are beneficial only in the presence of selection for those particular substances. In their absence the plasmid-carrying bacteria has a reduced growth rate due to the metabolic cost of maintaining the plasmid. Thus a tradeoff exists between benefit and burden resulting in persistence or loss of the plasmid as described by Lilley and Bailey (1997).
To study this phenomenon of plasmid persistence in heterogeneous environments, the authors investigated the effects of spatially heterogeneous Hg pollution on selection for P. fluorescens SBW25 carrying the Hg(r) plasmid pQBR103 relative to the plasmid-free strain.
1) The authors labeled the plasmid-free SBW25 chromosome with an RFP cassette and the plasmid pQBR103 with a GFP cassette.
2) They calculated maximum specific growth rates in liquid cultures of SBW25::rif and SBW25 (pQBR103::gfp).
3) Liquid monocultures of SBW25::rif and SBW25 (pQBR103::gfp) were prepared. Cells were harvested and resuspended in PBS (O.D=0.5). Mixtures having 1:1 ratio of Hg(r)-to-Hg(s) were diluted in PBS (to approximately 2 x 104 CFU /ml) and used to inoculate membranes.
4) Black Isopore polycarbonate membrane filters were pre-washed with sterile distilled water (SDW). The filters were incubated with inoculum on 0.7 R2A for 3 days at 21-23°C.
5) For heterogeneous treatment, the authors soaked 5 gm of carboxymethyl cellulose fibers with either 30 ml SDW or 1.15 or 7.66 mM HgCl(2) to have a final concentration of 0, 1,875 or 12,500 μg HgCl(2) g / cellulose. The cellulose mixtures were dried and crushed to separate the fibers. For random distribution of Hg foci, the cellulose fibers were sprayed down a sealed pressurized container onto the pre-grown bacterial culture on the membrane filter.
6) For uniform treatment, the authors placed the membranes on R2A supplemented with HgCl(2) to give a final concentration of 0, 2.5, 5 or 7.5 μM.
7) Visualization of the colonies was done by using an Eclipse E600 microscope. Images of at least 10 fields of view were captured.
8) Calculation of a(Hg)(r) gives the area of each field of view (FOV) occupied by Hg(r) relative to Hg(s) ( where Hg(r) is strain resistant to Hg and Hg(s) is strain sensitive to Hg) . W(Hg)(r) which is the relative fitness of Hg(r) populations was also calculated.
The results showed that when starting with 1:1 ratio of SBW25::rif and SBW25 (pQBR103::gfp), in the absence of Hg, a(Hg)(r) was 0.33 and W(Hg)(r) was 0.78 where mean area of FOVS occupied by Hg(s) was 74.42%. In the presence of uniform and heterogeneous Hg, a(Hg)(r) was found to increase with Hg concentration. However the value of W(Hg)(r) which is calculated over the entire population, increased only for the uniform Hg treatment and not for the heterogeneous treatment. They explain this by localized selection for Hg(r) in the heterogeneous condition, which did not increase the value of W(Hg)(r) considerably. To test this, they repeated the experiment with either a heterogeneous treatment of 9,375 μg HgCl(2) g/cellulose or a no-Hg control treatment and a variety of starting ratios of Hg(r) to Hg(s) bacteria. Changes in a(Hg)(r) between no-Hg control and heterogeneous treatments were proportional to the starting inoculum of Hg(r) bacteria. Starting with a smaller inoculum of Hg(r) resulted in a greater increase in selection. This lead them to conclude that negative frequency-dependent selection for the plasmid carrying bacteria was taking place in heterogeneously distributed spatial environments. Thus when the number of Hg(r) cells is low there is a higher selection for Hg(r) bacteria than when the number of Hg(r) cells is high.
Their conclusion supports previous work by Ellis, R. J., et al (2007) that demonstrated negative frequency-dependent selection in structured and unstructured environments under uniform Hg conditions. This study goes a step ahead and proves that negative frequency-dependent selection is taking place when Hg is distributed heterogeneously in a spatial environment. In order to determine the time periods over which coexistence of plasmid-bearing and plasmid-free cells takes place and other variables, the authors indicate that further work is to be carried out.
This paper is important because the authors carry out experimental work to demonstrate plasmid persistence in heterogeneous environments, which would be more typical of the “real-world” growth conditions for bacteria, rather than the usually more homogeneous laboratory conditions. A novel method was developed for creating a spatially heterogeneous environment using cellulose fibers imbued with HgCl(2) sprayed onto preinoculated membranes.
Primary Article
Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads
Additional references:
Rainey, P.B., and M. Travisano. 1998. Adaptive radiation in a heterogeneous environment. Nature 394:69-72.
Ellis, R. J., A. K. Lilley, S.J. Lacey, D. Murrell, and H.C.J. Godfray.2007.
Frequency-dependent advantages of plasmid carriage by Pseudomonas sp in homogeneous and spatially structured environments. ISME J. 1:92-95
Lilley, A.K., and M.J. Bailey.1997. Impact of plasmid pQBR103 acquisition and carriage on the phytosphere fitness of Pseudomonas fluorescens SBW25: burden and benefit. Appl. Environ. Microbiol.63:1584-1587.
Diya Sen, Graduate Student
University of Idaho
Monday, August 25, 2008
Friday, August 8, 2008
Acclimation of Subsurface Microbial Communities to Mercury
De Lipthay, J.R., Rasmussen, L.D., Oregaard, G., Simonsen, K., Bahl, M.I., Kroer, N., & Sørensen, S.J. (2008). FEMS Microbiology Ecology, 65, 145-155.
As contamination of our oceans, freshwater sources, and soils continues to be an increasing concern, the need for research addressing the potential of remediating contaminated environments also becomes increasingly evident. Mercury (Hg) contamination of soil and water is a particular concern, since mercury is toxic even at very low concentrations. In this article by Lipthay, et al., the authors studied the adaptation of microbial communities to mercury in both mercury-contaminated and non-contaminated subsurface soils. The findings in this study point to the importance of the role of plasmids in the acclimation to mercury of the soil bacteria communities and, on the other hand, their importance in bioremediation efforts.
The authors’ analysis of contaminated and non-contaminated soils included diverse microbiological techniques:
(1) the appearance of colonies on R2A agar plates with or without Hg(II) amendment (to test the abundance of culturable bacteria),
(2) substrate utilization patterns in Biolog ECOplates with or without Hg(II) amendment (to measure the ability of soil bacteria to utilize single carbon sources),
(3) substrate utilization patterns in Biolog mt-2 plates amended with increasing concentrations of Hg(II) (to estimate the minimal inhibitory concentration of Hg(II) in the soil communities),
(4) merA PCR (to test for the presence of merA genes in whole community soil DNA extracts),
(5) IncP trfA1 PCR (to test for the presence of trfA1 genes of the IncP-1 incompatibility group of plasmids in whole community soil DNA extracts), and
(6) 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis (to analyze the genetic diversity of the various bacterial soil communities).
As expected, the abundance of culturable, mercury-resistant bacteria was higher in the mercury-contaminated soils than in the non-contaminated soils and the soil communities from the contaminated site were better adapted to mercury. Results also suggested a higher diversity of mercury-resistant bacteria in the surface soil communities than in the subsurface soil communities. Treatment of the soils with mercury though, resulted in an equally diverse mercury-resistant population in both subsurface and surface soil communities. This increase in diversity following mercury “stimulation” is similar to results found in another study (Muller et al., 2001a), and the authors point out that it is probably the result of either 1) growth of previously low abundance resistant bacteria or 2) transfer of the mer operon by horizontal gene exchange, leading to elevated mercury resistance in the bacterial soil community.
The mercury tolerance of the soil bacteria communities decreased with depth in both the contaminated and non-contaminated soils, with tolerance levels being greater overall in microbial communities from the contaminated soils. Mercury tolerance increased significantly after mercury amendment, indicating a shift in community composition. This increase in tolerance is also most likely due to horizontal gene transfer of the mer operon. Before mercury was added to soils, PCR targeting merA genes resulted only in PCR products from the contaminated soils (cultivation-based methods were used to show that mercury-resistant bacteria were present in non-contaminated soils). After mercury-amendment merA genes were found in all soils, thus demonstrating the adaptive potential of the soil bacteria communities.
IncP-1 trfA1 genes were detected only in DNA extracted from the contaminated soils, not from the non-contaminated soils, and were also more abundant in the surface soil than in subsurface soils.
Another interesting finding of this study to note here is that the microbial diversity did not generally decrease with soil depth, thus contradicting one of the authors’ original hypotheses.
Through biparental exogenous plasmid isolation, the authors found four different mercury-resistance plasmids (pCPH-001, -002, -003, and -004). Further analysis of these plasmids confirmed that all four had identical partial trfA sequences (and belonged to the IncP-1B group), yet had different merA genes. All four plasmid types were isolated from the contaminated surface soil; however, only plasmid pCPH-001 was isolated from the two subsurface soils. This finding of a higher diversity of mercury-resistance plasmids in the surface soil compared with the subsurface soils corresponds well with the findings of a higher diversity of mercury-resistant bacteria in the contaminated surface soil. In contrast, no mercury-resistance plasmids were retrieved from the non-contaminated soils. The observations in this study, that IncP-1 plasmids are present in contaminated but not in non-contaminated soils, are similar to those in other studies (Smalla et al., 2000; Campbell et al., 1995) and are evidence of the importance of IncP-1 plasmids in the dissemination of adaptive functional traits in microbial communities.
The authors suggest that the findings of this study are important to bear in mind when considering bioremediation of mercury-polluted environments.
Additional References:
Campbell, J.I.A., Jacobsen, C.S., & Sørensen, J. (1995). Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiology Ecology, 18, 51–62.
Muller, A.K., Rasmussen, L.D., & Sørensen, S.J. (2001a). Adaptation of the bacterial community to mercury contamination. FEMS Microbiology Letters, 204, 49–53.
Smalla, K., Krogerrecklenfort, E., Heuer, H., et al. (2000) PCR-based detection of mobile genetic elements in total community DNA. Microbiology, 146, 1256–1257.
Matt Bauer, B.S.
University of Idaho
De Lipthay, J.R., Rasmussen, L.D., Oregaard, G., Simonsen, K., Bahl, M.I., Kroer, N., & Sørensen, S.J. (2008). FEMS Microbiology Ecology, 65, 145-155.
As contamination of our oceans, freshwater sources, and soils continues to be an increasing concern, the need for research addressing the potential of remediating contaminated environments also becomes increasingly evident. Mercury (Hg) contamination of soil and water is a particular concern, since mercury is toxic even at very low concentrations. In this article by Lipthay, et al., the authors studied the adaptation of microbial communities to mercury in both mercury-contaminated and non-contaminated subsurface soils. The findings in this study point to the importance of the role of plasmids in the acclimation to mercury of the soil bacteria communities and, on the other hand, their importance in bioremediation efforts.
The authors’ analysis of contaminated and non-contaminated soils included diverse microbiological techniques:
(1) the appearance of colonies on R2A agar plates with or without Hg(II) amendment (to test the abundance of culturable bacteria),
(2) substrate utilization patterns in Biolog ECOplates with or without Hg(II) amendment (to measure the ability of soil bacteria to utilize single carbon sources),
(3) substrate utilization patterns in Biolog mt-2 plates amended with increasing concentrations of Hg(II) (to estimate the minimal inhibitory concentration of Hg(II) in the soil communities),
(4) merA PCR (to test for the presence of merA genes in whole community soil DNA extracts),
(5) IncP trfA1 PCR (to test for the presence of trfA1 genes of the IncP-1 incompatibility group of plasmids in whole community soil DNA extracts), and
(6) 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis (to analyze the genetic diversity of the various bacterial soil communities).
As expected, the abundance of culturable, mercury-resistant bacteria was higher in the mercury-contaminated soils than in the non-contaminated soils and the soil communities from the contaminated site were better adapted to mercury. Results also suggested a higher diversity of mercury-resistant bacteria in the surface soil communities than in the subsurface soil communities. Treatment of the soils with mercury though, resulted in an equally diverse mercury-resistant population in both subsurface and surface soil communities. This increase in diversity following mercury “stimulation” is similar to results found in another study (Muller et al., 2001a), and the authors point out that it is probably the result of either 1) growth of previously low abundance resistant bacteria or 2) transfer of the mer operon by horizontal gene exchange, leading to elevated mercury resistance in the bacterial soil community.
The mercury tolerance of the soil bacteria communities decreased with depth in both the contaminated and non-contaminated soils, with tolerance levels being greater overall in microbial communities from the contaminated soils. Mercury tolerance increased significantly after mercury amendment, indicating a shift in community composition. This increase in tolerance is also most likely due to horizontal gene transfer of the mer operon. Before mercury was added to soils, PCR targeting merA genes resulted only in PCR products from the contaminated soils (cultivation-based methods were used to show that mercury-resistant bacteria were present in non-contaminated soils). After mercury-amendment merA genes were found in all soils, thus demonstrating the adaptive potential of the soil bacteria communities.
IncP-1 trfA1 genes were detected only in DNA extracted from the contaminated soils, not from the non-contaminated soils, and were also more abundant in the surface soil than in subsurface soils.
Another interesting finding of this study to note here is that the microbial diversity did not generally decrease with soil depth, thus contradicting one of the authors’ original hypotheses.
Through biparental exogenous plasmid isolation, the authors found four different mercury-resistance plasmids (pCPH-001, -002, -003, and -004). Further analysis of these plasmids confirmed that all four had identical partial trfA sequences (and belonged to the IncP-1B group), yet had different merA genes. All four plasmid types were isolated from the contaminated surface soil; however, only plasmid pCPH-001 was isolated from the two subsurface soils. This finding of a higher diversity of mercury-resistance plasmids in the surface soil compared with the subsurface soils corresponds well with the findings of a higher diversity of mercury-resistant bacteria in the contaminated surface soil. In contrast, no mercury-resistance plasmids were retrieved from the non-contaminated soils. The observations in this study, that IncP-1 plasmids are present in contaminated but not in non-contaminated soils, are similar to those in other studies (Smalla et al., 2000; Campbell et al., 1995) and are evidence of the importance of IncP-1 plasmids in the dissemination of adaptive functional traits in microbial communities.
The authors suggest that the findings of this study are important to bear in mind when considering bioremediation of mercury-polluted environments.
Additional References:
Campbell, J.I.A., Jacobsen, C.S., & Sørensen, J. (1995). Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiology Ecology, 18, 51–62.
Muller, A.K., Rasmussen, L.D., & Sørensen, S.J. (2001a). Adaptation of the bacterial community to mercury contamination. FEMS Microbiology Letters, 204, 49–53.
Smalla, K., Krogerrecklenfort, E., Heuer, H., et al. (2000) PCR-based detection of mobile genetic elements in total community DNA. Microbiology, 146, 1256–1257.
Matt Bauer, B.S.
University of Idaho
Friday, July 25, 2008
The Role of Plasmid-encoded H-NS-like Protein
"An H-NS-like stealth protein aids horizontal DNA transmission in bacteria" Doyle M et al., Science 315: 251-252 (2007)
H-NS is one of the abundant DNA-binding proteins that are found in gram-negative bacterial cells. H-NS is known to bind to A+T-rich sequences and regulate expression of a large number of chromosomal genes (Fang FC and Rimsky S, 2008) . Recently, it has become clear that some narrow-host-range plasmids derived from gram-negative bacteria encode H-NS-like proteins. Yet, their roles were still obscure.
So far, it has been shown that Sfh, an H-NS paralog encoded by IncHI1 group plasmid pSf-R27, interacts with host H-NS, and both proteins are functionally exchangeable (Deighan P et al., 2003; Beloin C et al., 2003). People thus might think that plasmid-encoded H-NS-like proteins influence global gene expression of host bacteria. Interestingly, the results shown in this article look to be in the contrary: the absence of Shf disturbed global gene expression of transconjugants. In this article, authors proposed that plasmid-encoded H-NS is a stealth protein that allows host bacteria to carry A+T-rich plasmids with minimal effect on global gene expression and "fitness", by preventing plasmids from titrating cellular pool of H-NS.
Authors introduced pSf-R27, with or without the sfh gene, from original host Shigella flexeneri into Salmonella Typhimurium, and analyzed the transcriptome as well as several phenotypes of transconjugants. Interestingly, the transfer of wild-type pSf-R27 resulted in a few change in the recipient, but transfer of pSf-27Δsfh resulted in the drastic changes in expression of a wide range of genes. Noteworthy phenotypes of the recipient carrying sfh mutant were increased resistance to UV, increased virulence (persistence in macrophage) and reduced motility. These phenotypes are reminiscent of the chromosomal hns mutant (Navarre WW et al., 2006). Authors then showed that the sfh mutation significantly reduced the fitness of recipient (this phenotype was completely complemented by supplying Sfh in trans from another plasmid). To figure out if the reduction of fitness resulted from the titration of "host" H-NS by A+T-rich sequence on the plasmid, authors constructed a pUC18 derivative that carried chromosome-derived A+T-rich DNA fragment and introduced it into the recipient cells, instead of pSf-R27Δsfh. The recipient that carries the pUC18 derivative caused reduction in the fitness, and this reduction was complemented in the presence of Sfh, as in the case of pSf-R27.
Based on these observations authors proposed that sfh is a "stealth" gene that allows the A+T-rich pSf-R27 to invade a new bacterial host with a minimal impact on global gene expression patterns and fitness. They added that the positive effects of sfh on the fitness can be applied to biotechnology to construct more stable cloning vectors.
PRIMARY ARTICLE
Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, Dorman CJ.
An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.
Science 2007, 315:251-2.
ADDITIONAL REFERENCES
Fang FC, Rimsky S.
New insights into transcriptional regulation by H-NS.
Curr. Opin. Microbiol. 2008, 11:113-20.
Deighan P, Beloin C, Dorman CJ.
Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.
Mol. Microbiol. 2003, 48:1401-16.
Beloin C, Deighan P, Doyle M, Dorman CJ.
Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.
Mol. Genet. Genomics 2003, 270:66-77.
Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC. Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella.
Science. 2006 Jul 14;313(5784):236-8.
Hirokazu Yano (Ph. D.)
University of Idaho
H-NS is one of the abundant DNA-binding proteins that are found in gram-negative bacterial cells. H-NS is known to bind to A+T-rich sequences and regulate expression of a large number of chromosomal genes (Fang FC and Rimsky S, 2008) . Recently, it has become clear that some narrow-host-range plasmids derived from gram-negative bacteria encode H-NS-like proteins. Yet, their roles were still obscure.
So far, it has been shown that Sfh, an H-NS paralog encoded by IncHI1 group plasmid pSf-R27, interacts with host H-NS, and both proteins are functionally exchangeable (Deighan P et al., 2003; Beloin C et al., 2003). People thus might think that plasmid-encoded H-NS-like proteins influence global gene expression of host bacteria. Interestingly, the results shown in this article look to be in the contrary: the absence of Shf disturbed global gene expression of transconjugants. In this article, authors proposed that plasmid-encoded H-NS is a stealth protein that allows host bacteria to carry A+T-rich plasmids with minimal effect on global gene expression and "fitness", by preventing plasmids from titrating cellular pool of H-NS.
Authors introduced pSf-R27, with or without the sfh gene, from original host Shigella flexeneri into Salmonella Typhimurium, and analyzed the transcriptome as well as several phenotypes of transconjugants. Interestingly, the transfer of wild-type pSf-R27 resulted in a few change in the recipient, but transfer of pSf-27Δsfh resulted in the drastic changes in expression of a wide range of genes. Noteworthy phenotypes of the recipient carrying sfh mutant were increased resistance to UV, increased virulence (persistence in macrophage) and reduced motility. These phenotypes are reminiscent of the chromosomal hns mutant (Navarre WW et al., 2006). Authors then showed that the sfh mutation significantly reduced the fitness of recipient (this phenotype was completely complemented by supplying Sfh in trans from another plasmid). To figure out if the reduction of fitness resulted from the titration of "host" H-NS by A+T-rich sequence on the plasmid, authors constructed a pUC18 derivative that carried chromosome-derived A+T-rich DNA fragment and introduced it into the recipient cells, instead of pSf-R27Δsfh. The recipient that carries the pUC18 derivative caused reduction in the fitness, and this reduction was complemented in the presence of Sfh, as in the case of pSf-R27.
Based on these observations authors proposed that sfh is a "stealth" gene that allows the A+T-rich pSf-R27 to invade a new bacterial host with a minimal impact on global gene expression patterns and fitness. They added that the positive effects of sfh on the fitness can be applied to biotechnology to construct more stable cloning vectors.
PRIMARY ARTICLE
Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, Dorman CJ.
An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.
Science 2007, 315:251-2.
ADDITIONAL REFERENCES
Fang FC, Rimsky S.
New insights into transcriptional regulation by H-NS.
Curr. Opin. Microbiol. 2008, 11:113-20.
Deighan P, Beloin C, Dorman CJ.
Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.
Mol. Microbiol. 2003, 48:1401-16.
Beloin C, Deighan P, Doyle M, Dorman CJ.
Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.
Mol. Genet. Genomics 2003, 270:66-77.
Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC. Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella.
Science. 2006 Jul 14;313(5784):236-8.
Hirokazu Yano (Ph. D.)
University of Idaho
Saturday, July 19, 2008
Direct Visualization of Plasmid Transfer
Direct evidence of extant in situ plasmid transfer in natural environments has typically been obtained by identifying plasmid-encoded phenotypes following the introduction of donor strains. Many plasmids do not encode any known functions (cryptic plasmids); in many unknown plasmids, functions are not determined or there is no easy method to select plasmid-containing cells. So there is the necessity to use known reporter markers, like antibiotic resistance, lacZ, gusA, luxAB or fluorescent proteins genes to label plasmids prior to study. The main advantage of using luciferase and especially fluorescent proteins is that it is possible to detect the presence of plasmid DNA without plating (non-culturable bacteria) and adding additional substrates to the environment. Using of fluorescent proteins enables direct visualization of plasmid transfer but has also some limitations. First is using epifluorescence microscopy or flow-cytometry-based method to detect fluorescent cells, both of which are technically demanding and require expensive equipment. The second limitation arises from the properties of fluorescent proteins. They need some time - from a few to several hours, to produce the strong, detectable signal. So this make impossible to detect precisely the time of plasmid DNA entering the recipient cell.
In the paper “Direct visualization of horizontal gene transfer” by Ana Babic et al. (2008) authors developed an experimental system that enables them to distinguish the transferred donor DNA from both donor and recipient DNA, and to visualize DNA transfer and recombination by means of fluorescence microscopy in real time, at the level of individual living cells. This tool also allowed them to quantify the ongoing transfer of DNA during conjugation and to acquire time-lapse movies that follow the fate of the newly acquired DNA in individual cells through any number of cell divisions.
This method uses fusion of YFP gene with seqA gene. This translational fusion driven from the native seqA promoter was introduced into E.coli chromosome replacing the wild type seqA allel. SeqA protein has strong affinity for DNA which is hemimethylated by Dam methylase at GATC sequences. Such a hemimethylated DNA usually occurs in the replication forks during replication and SeqA-YFP fusion protein had been found bound to chromosomal DNA previously. When the host strain lacks Dam methylase, the chromosomal DNA is not methylated and SeqA-YFP protein is dispersed in the cytoplasm giving dim background fluorescence.
During conjugation single stranded DNA is transferred from a donor strain to the recipient cells and the second DNA strand is synthesized. When plasmid DNA is methylated by Dam methylase and transferred to a Dam deficient strain, stable hemimethylated duplex is formed. Such a duplex is recognized by SeqA-YFP fusion protein and gives strong fluorescence foci.
Using this technique authors were able to detect presence of transferred plasmid DNA in transconjugants as quickly as 5 minutes after mixing together parental strains. After 30-40 minutes almost all recipient cells in the vicinity of donors showed fluorescent foci. In comparison, the RFP protein expressed from the transferred plasmid from tetracycline promoter showed visible signal 2 hours after transfer. They also showed that in the case of F plasmid direct cell wall contact is not necessary for transfer; this means that single stranded plasmid DNA is transferred from cell to cell thru the sex pili.
To summarize this method allows them to visualize and quantify the DNA of any sequence as it is being transferred from one individual cell to another, and to watch its stable genomic acquisition via genetic recombination (horizontal gene transfer) in real time. This experimental system can be applied to monitor horizontal gene transfer by indefinitely following the fate of DNA acquired in intra- and interspecies crosses.
PRIMARY ARTICLE:
Ana Babic, Ariel B. Lindner, Marin Vuli, Eric J. Stewart and Miroslav Radman 2008, Direct Visualization of Horizontal Gene Transfer. Science 319, pp. 1533 - 1536.
In the paper “Direct visualization of horizontal gene transfer” by Ana Babic et al. (2008) authors developed an experimental system that enables them to distinguish the transferred donor DNA from both donor and recipient DNA, and to visualize DNA transfer and recombination by means of fluorescence microscopy in real time, at the level of individual living cells. This tool also allowed them to quantify the ongoing transfer of DNA during conjugation and to acquire time-lapse movies that follow the fate of the newly acquired DNA in individual cells through any number of cell divisions.
This method uses fusion of YFP gene with seqA gene. This translational fusion driven from the native seqA promoter was introduced into E.coli chromosome replacing the wild type seqA allel. SeqA protein has strong affinity for DNA which is hemimethylated by Dam methylase at GATC sequences. Such a hemimethylated DNA usually occurs in the replication forks during replication and SeqA-YFP fusion protein had been found bound to chromosomal DNA previously. When the host strain lacks Dam methylase, the chromosomal DNA is not methylated and SeqA-YFP protein is dispersed in the cytoplasm giving dim background fluorescence.
During conjugation single stranded DNA is transferred from a donor strain to the recipient cells and the second DNA strand is synthesized. When plasmid DNA is methylated by Dam methylase and transferred to a Dam deficient strain, stable hemimethylated duplex is formed. Such a duplex is recognized by SeqA-YFP fusion protein and gives strong fluorescence foci.
Using this technique authors were able to detect presence of transferred plasmid DNA in transconjugants as quickly as 5 minutes after mixing together parental strains. After 30-40 minutes almost all recipient cells in the vicinity of donors showed fluorescent foci. In comparison, the RFP protein expressed from the transferred plasmid from tetracycline promoter showed visible signal 2 hours after transfer. They also showed that in the case of F plasmid direct cell wall contact is not necessary for transfer; this means that single stranded plasmid DNA is transferred from cell to cell thru the sex pili.
To summarize this method allows them to visualize and quantify the DNA of any sequence as it is being transferred from one individual cell to another, and to watch its stable genomic acquisition via genetic recombination (horizontal gene transfer) in real time. This experimental system can be applied to monitor horizontal gene transfer by indefinitely following the fate of DNA acquired in intra- and interspecies crosses.
PRIMARY ARTICLE:
Ana Babic, Ariel B. Lindner, Marin Vuli, Eric J. Stewart and Miroslav Radman 2008, Direct Visualization of Horizontal Gene Transfer. Science 319, pp. 1533 - 1536.
ADDITIONAL REFERENCES:
Søren J. Sørensen, Mark Bailey, Lars H. Hansen, Niels Kroer and Stefan Wuertz 2005, Studing Plasmid Horizontal Transfer In Situ: A Critical Review. Nature Rev. 3, pp.700 - 710.
Sota Hiraga, Chiyome Ichinose, Hironori Niki and Mitsuyoshi Yamazoe 1998, Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes in E. coli. Mol. Cell 1, pp.381 - 397.
Dr Jaroslaw E. Krol
University of Idaho
F-A
Sunday, July 13, 2008
Reticulate classification of mobile genetic elements
“Reticulate representation of evolutionary and functional relationships between phage genomes.” by Lima-Mendez et al. (2008)
In this paper, the authors note that mobile genetic elements (MGE) in prokaryotes (such as phages, plasmids, conjugative transposons, and genomic islands) show mosaic structures, indicating the importance of horizontal gene exchange in their evolution. These elements represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification schemes cannot be applied to these genetic elements in part due to the intrinsic inability of tree-based methods to efficiently deal with mosaicism.
To solve the problem, Lima-Mendez et al. (2008) proposed a framework for a reticulate classification of phages based on gene content; i.e., presence (1) or absence (0) of protein family. First, the authors built a graph, where nodes represent phages and lines represent similarities in gene content between phages. Then, the authors applied a two-step clustering [Markov clustering (MCL) and fuzzy clustering] to this graph to generate a reticulate classification of phages: each phage is represented by a membership vector, which quantitatively characterizes its membership in the set of clusters. Phages within the same MCL cluster are likely descendant from a unique module combination, and one phage could belong to several clusters (Lawrence et al. 2002); for example, phage lambda belongs almost equally to two different clusters. Lima-Mendez et al. (2008) stated that “The weight of the intracluster connections represents 79% of the total weight of the connections of the network. This number can be taken as a rough estimate of the contribution of vertical evolution in this network. However, phages from different MCL clusters may be also be related through vertical evolution, but they might have diverged so much that sequence similarities are no longer recognizable or only some [evolutionary cohesive] modules may have been vertically inherited, whereas others have been replaced through horizontal gene transfer.” Thus, it is still difficult to estimate the contribution of different evolutionary events (i.e., vertical and horizontal gene transfer). Kunin and Ouzounis (2003) suggested a framework for the inference of presence or absence of individual protein families at any node on a phylogenetic tree, and assumed that: (1) A protein family shared by most of the clade members would be vertically transmitted; (2) If a protein family is present in most of the descendants of a particular ancestor, but is not found in some subclade, the observed gene absence would normally result from gene loss; and (3) A protein family interspersed across distantly related clades would be horizontally transferred. This assumption cannot detect horizontal gene transfer (HGT) among closely related species, as is true for most methods used to identify HGT (those based on phylogenetic information and compositional features). However, it is well recognized that phage and plasmid transfers (and consequently HGT) should be more likely among closely related species than among distantly related species.
Phylogenetic profiles have been widely applied to bacterial genomes to predict functional links between proteins on the assumption that proteins interacting in metabolic pathways or physical structure would be required to co-occur in genomes (Pellegrini et al. 1999). Lima-Mendez et al. (2008) clustered genes based on their “phylogenetic profiles” to define “evolutionary cohesive modules.” In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules, suggesting that the phylogenetic profile method does not work well at predicting protein function in virulent phages. The Lima-Mendez analysis reminds us that we must be careful to consider the total context of the MGE, and not only the genome content.
The Lima-Mendez analysis was implemented using Network Analysis Tools (NeAT) (Brohée et al. 2008), available at http://rsat.ulb.ac.be/rsat/index_neat.html.
PRIMARY ARTICLE:
Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.
ADDITIONAL REFERENCES:
Lawrence JG, Hatfull GF, Hendrix RW. J Bacteriol. (2002) 184:4891-905. Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches.
Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.
Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.
Brohée S, Faust K, Lima-Mendez G, Sand O, Janky R, Vanderstocken G, Deville Y, van Helden J. Nucleic Acids Res. (2008) 36(Web Server issue):W444-51. NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.
Dr. Haruo Suzuki
University of Idaho
In this paper, the authors note that mobile genetic elements (MGE) in prokaryotes (such as phages, plasmids, conjugative transposons, and genomic islands) show mosaic structures, indicating the importance of horizontal gene exchange in their evolution. These elements represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification schemes cannot be applied to these genetic elements in part due to the intrinsic inability of tree-based methods to efficiently deal with mosaicism.
To solve the problem, Lima-Mendez et al. (2008) proposed a framework for a reticulate classification of phages based on gene content; i.e., presence (1) or absence (0) of protein family. First, the authors built a graph, where nodes represent phages and lines represent similarities in gene content between phages. Then, the authors applied a two-step clustering [Markov clustering (MCL) and fuzzy clustering] to this graph to generate a reticulate classification of phages: each phage is represented by a membership vector, which quantitatively characterizes its membership in the set of clusters. Phages within the same MCL cluster are likely descendant from a unique module combination, and one phage could belong to several clusters (Lawrence et al. 2002); for example, phage lambda belongs almost equally to two different clusters. Lima-Mendez et al. (2008) stated that “The weight of the intracluster connections represents 79% of the total weight of the connections of the network. This number can be taken as a rough estimate of the contribution of vertical evolution in this network. However, phages from different MCL clusters may be also be related through vertical evolution, but they might have diverged so much that sequence similarities are no longer recognizable or only some [evolutionary cohesive] modules may have been vertically inherited, whereas others have been replaced through horizontal gene transfer.” Thus, it is still difficult to estimate the contribution of different evolutionary events (i.e., vertical and horizontal gene transfer). Kunin and Ouzounis (2003) suggested a framework for the inference of presence or absence of individual protein families at any node on a phylogenetic tree, and assumed that: (1) A protein family shared by most of the clade members would be vertically transmitted; (2) If a protein family is present in most of the descendants of a particular ancestor, but is not found in some subclade, the observed gene absence would normally result from gene loss; and (3) A protein family interspersed across distantly related clades would be horizontally transferred. This assumption cannot detect horizontal gene transfer (HGT) among closely related species, as is true for most methods used to identify HGT (those based on phylogenetic information and compositional features). However, it is well recognized that phage and plasmid transfers (and consequently HGT) should be more likely among closely related species than among distantly related species.
Phylogenetic profiles have been widely applied to bacterial genomes to predict functional links between proteins on the assumption that proteins interacting in metabolic pathways or physical structure would be required to co-occur in genomes (Pellegrini et al. 1999). Lima-Mendez et al. (2008) clustered genes based on their “phylogenetic profiles” to define “evolutionary cohesive modules.” In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules, suggesting that the phylogenetic profile method does not work well at predicting protein function in virulent phages. The Lima-Mendez analysis reminds us that we must be careful to consider the total context of the MGE, and not only the genome content.
The Lima-Mendez analysis was implemented using Network Analysis Tools (NeAT) (Brohée et al. 2008), available at http://rsat.ulb.ac.be/rsat/index_neat.html.
PRIMARY ARTICLE:
Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.
ADDITIONAL REFERENCES:
Lawrence JG, Hatfull GF, Hendrix RW. J Bacteriol. (2002) 184:4891-905. Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches.
Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.
Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.
Brohée S, Faust K, Lima-Mendez G, Sand O, Janky R, Vanderstocken G, Deville Y, van Helden J. Nucleic Acids Res. (2008) 36(Web Server issue):W444-51. NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.
Dr. Haruo Suzuki
University of Idaho
Friday, July 11, 2008
Introduction to this blog
There is no doubt that bacteria evolve and adapt to local environments in part by horizontal (or lateral) gene transfer between closely and very distantly related organisms. The detection of very similar genes in distinct bacteria showed that large fractions of bacterial genomes have arisen through horizontal gene transfer. Consistent with this, studies done in the laboratory and in the field have shown that the horizontal transfer of genes between bacterial populations readily occurs in various habitats. Thus bacteria have access to a common pool of genes, called the ‘virtual genome’ (VG) or horizontal gene pool (‘HGP’). This sharing of genes from the VG allows ‘wholesale’ acquisition of useful traits such as drug resistance, heavy metal resistance, pollutant degradation, virulence factors, and many more.
Of the various MGEs that play a role in genetic exchange among bacteria, plasmids are of particular interest because many are self-transferable and able to replicate in a wide range of hosts. They often carry antibiotic resistance, pollutant degradation or other provide a fitness advantage to their host. Because plasmids can transfer among different bacterial species, they play an important role in the ability of bacteria to degrade environmental contaminants and to become resistant to drugs used to treat infectious diseases of plants, animals and humans. However, little is known about the genetic structure of these mobile elements and the full range of functions that they encode. To gain insight into the issues outlined above, our plasmid genome sequencing project is analyzing the genome sequences of 100 plasmids that have a broad host-range (also called BHR plasmids). The sequenced plasmids were obtained from soil, water, and sewage sludge samples from around the globe. The project includes finishing the sequencing and annotation of the plasmids, and then interpreting the sequence information to better understand the evolutionary history of plasmids, and their role in bacterial chromosome evolution and adaptation to new environments. The information collected during this project will add significant new data to the paltry plasmid sequence database that now exists, which is also skewed towards plasmids relevant to human infectious diseases.
With this blog we hope to spark your interest in horizontal gene transfer among bacteria, and the diversity, ecology and evolution of bacterial plasmids, and generate discussion about the latest findings in the field.
Dr. Eva Top
University of Idaho
Of the various MGEs that play a role in genetic exchange among bacteria, plasmids are of particular interest because many are self-transferable and able to replicate in a wide range of hosts. They often carry antibiotic resistance, pollutant degradation or other provide a fitness advantage to their host. Because plasmids can transfer among different bacterial species, they play an important role in the ability of bacteria to degrade environmental contaminants and to become resistant to drugs used to treat infectious diseases of plants, animals and humans. However, little is known about the genetic structure of these mobile elements and the full range of functions that they encode. To gain insight into the issues outlined above, our plasmid genome sequencing project is analyzing the genome sequences of 100 plasmids that have a broad host-range (also called BHR plasmids). The sequenced plasmids were obtained from soil, water, and sewage sludge samples from around the globe. The project includes finishing the sequencing and annotation of the plasmids, and then interpreting the sequence information to better understand the evolutionary history of plasmids, and their role in bacterial chromosome evolution and adaptation to new environments. The information collected during this project will add significant new data to the paltry plasmid sequence database that now exists, which is also skewed towards plasmids relevant to human infectious diseases.
With this blog we hope to spark your interest in horizontal gene transfer among bacteria, and the diversity, ecology and evolution of bacterial plasmids, and generate discussion about the latest findings in the field.
Dr. Eva Top
University of Idaho
Subscribe to:
Posts (Atom)