Slater, Frances R.; Bruce, Kenneth D.; Ellis, Richard J.; Lilley, Andrew K.; Turner, Sarah L.
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.
Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads
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