Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads

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

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