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Novel assay to measure the plasmid mobilizing potential of mixed microbial communities.

Klümper U, Droumpali A, Dechesne A, Smets BF - Front Microbiol (2014)

Bottom Line: The permissiveness of the model community for RP4 [at 1.16 × 10(-4) transconjugants per recipient (T/R)] was similar to that previously measured for soil microbial communities.RSF1010 was mobilized by the model community at a frequency of 1.16 × 10(-5) T/R, only one order of magnitude lower than its permissiveness to RP4.This method has the potential to provide such insights; in addition it allows for the direct isolation of in situ mobilizing plasmids together with their endogenous hosts.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Engineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We compare the transfer frequency of a mobilizable plasmid to that of a mobilizing and conjugal plasmid measured for a model strain and for the assayed community. With Pseudomonas putida carrying the gfp-tagged mobilizable IncQ plasmid RSF1010 as donor strain, we conducted solid surface mating experiments with either a P. putida strain carrying the mobilizing IncP-1α plasmid RP4 or a model bacterial community that was extracted from the inner walls of a domestic shower conduit. Additionally, we estimated the permissiveness of the same community for RP4 using P. putida as donor strain. The permissiveness of the model community for RP4 [at 1.16 × 10(-4) transconjugants per recipient (T/R)] was similar to that previously measured for soil microbial communities. RSF1010 was mobilized by the model community at a frequency of 1.16 × 10(-5) T/R, only one order of magnitude lower than its permissiveness to RP4. This mobilization frequency is unexpectedly high considering that (i) mobilization requires the presence of mobilizing conjugal plasmids within the permissive fraction of the recipients; (ii) in pure culture experiments with P. putida retromobilization of RSF1010 through RP4 only took place in approximately half of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities. This method has the potential to provide such insights; in addition it allows for the direct isolation of in situ mobilizing plasmids together with their endogenous hosts.

No MeSH data available.


Related in: MedlinePlus

Detection and quantification of transconjugant microcolonies by fluorescent microscopy. Fluorescence based stereomicroscopic images and image analysis of an example filter mating of Pseudomonas putida KT2440::lacIq-Lpp-mCherry-KmR (RSF1010::gfp) with the recipient community. (A) corresponds to the red fluorescent channel, displaying donor microcolonies. (B) shows the green fluorescent channel, corresponding to the transconjugal microcolonies that received the plasmid through retromobilization. (C) is a composite image of both channels with increased contrasts. Transconjugal microcolonies can be found in direct proximity to donor colonies. (D) illustrates counting of transconjugal colonies through a macro that increases contrast of the images, subtracts background, eliminates the poorly illuminated corners and counts green fluorescent object larger than 4 μm2.
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Figure 3: Detection and quantification of transconjugant microcolonies by fluorescent microscopy. Fluorescence based stereomicroscopic images and image analysis of an example filter mating of Pseudomonas putida KT2440::lacIq-Lpp-mCherry-KmR (RSF1010::gfp) with the recipient community. (A) corresponds to the red fluorescent channel, displaying donor microcolonies. (B) shows the green fluorescent channel, corresponding to the transconjugal microcolonies that received the plasmid through retromobilization. (C) is a composite image of both channels with increased contrasts. Transconjugal microcolonies can be found in direct proximity to donor colonies. (D) illustrates counting of transconjugal colonies through a macro that increases contrast of the images, subtracts background, eliminates the poorly illuminated corners and counts green fluorescent object larger than 4 μm2.

Mentions: Quantification of transfer events was performed with a custom-made macro written in Image Pro Plus 7.1. This macro successively extracts and subtracts the background from the original image, performs a best-fit equalization of the image intensity, before detecting bright objects larger than 4 μm2 based on automatic segmentation. Analysis of images was limited to the brightly illuminated elliptic central area of the field of view (Figure 3). All images were manually controlled for enumeration errors, and values corrected if deviations were noted. The number of gfp-positive colonies (transfer events) detected was scaled up to the total filter area and transfer frequency was calculated by dividing this number by the number of potential recipients originally placed on the filter.


Novel assay to measure the plasmid mobilizing potential of mixed microbial communities.

Klümper U, Droumpali A, Dechesne A, Smets BF - Front Microbiol (2014)

Detection and quantification of transconjugant microcolonies by fluorescent microscopy. Fluorescence based stereomicroscopic images and image analysis of an example filter mating of Pseudomonas putida KT2440::lacIq-Lpp-mCherry-KmR (RSF1010::gfp) with the recipient community. (A) corresponds to the red fluorescent channel, displaying donor microcolonies. (B) shows the green fluorescent channel, corresponding to the transconjugal microcolonies that received the plasmid through retromobilization. (C) is a composite image of both channels with increased contrasts. Transconjugal microcolonies can be found in direct proximity to donor colonies. (D) illustrates counting of transconjugal colonies through a macro that increases contrast of the images, subtracts background, eliminates the poorly illuminated corners and counts green fluorescent object larger than 4 μm2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273639&req=5

Figure 3: Detection and quantification of transconjugant microcolonies by fluorescent microscopy. Fluorescence based stereomicroscopic images and image analysis of an example filter mating of Pseudomonas putida KT2440::lacIq-Lpp-mCherry-KmR (RSF1010::gfp) with the recipient community. (A) corresponds to the red fluorescent channel, displaying donor microcolonies. (B) shows the green fluorescent channel, corresponding to the transconjugal microcolonies that received the plasmid through retromobilization. (C) is a composite image of both channels with increased contrasts. Transconjugal microcolonies can be found in direct proximity to donor colonies. (D) illustrates counting of transconjugal colonies through a macro that increases contrast of the images, subtracts background, eliminates the poorly illuminated corners and counts green fluorescent object larger than 4 μm2.
Mentions: Quantification of transfer events was performed with a custom-made macro written in Image Pro Plus 7.1. This macro successively extracts and subtracts the background from the original image, performs a best-fit equalization of the image intensity, before detecting bright objects larger than 4 μm2 based on automatic segmentation. Analysis of images was limited to the brightly illuminated elliptic central area of the field of view (Figure 3). All images were manually controlled for enumeration errors, and values corrected if deviations were noted. The number of gfp-positive colonies (transfer events) detected was scaled up to the total filter area and transfer frequency was calculated by dividing this number by the number of potential recipients originally placed on the filter.

Bottom Line: The permissiveness of the model community for RP4 [at 1.16 × 10(-4) transconjugants per recipient (T/R)] was similar to that previously measured for soil microbial communities.RSF1010 was mobilized by the model community at a frequency of 1.16 × 10(-5) T/R, only one order of magnitude lower than its permissiveness to RP4.This method has the potential to provide such insights; in addition it allows for the direct isolation of in situ mobilizing plasmids together with their endogenous hosts.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Engineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We compare the transfer frequency of a mobilizable plasmid to that of a mobilizing and conjugal plasmid measured for a model strain and for the assayed community. With Pseudomonas putida carrying the gfp-tagged mobilizable IncQ plasmid RSF1010 as donor strain, we conducted solid surface mating experiments with either a P. putida strain carrying the mobilizing IncP-1α plasmid RP4 or a model bacterial community that was extracted from the inner walls of a domestic shower conduit. Additionally, we estimated the permissiveness of the same community for RP4 using P. putida as donor strain. The permissiveness of the model community for RP4 [at 1.16 × 10(-4) transconjugants per recipient (T/R)] was similar to that previously measured for soil microbial communities. RSF1010 was mobilized by the model community at a frequency of 1.16 × 10(-5) T/R, only one order of magnitude lower than its permissiveness to RP4. This mobilization frequency is unexpectedly high considering that (i) mobilization requires the presence of mobilizing conjugal plasmids within the permissive fraction of the recipients; (ii) in pure culture experiments with P. putida retromobilization of RSF1010 through RP4 only took place in approximately half of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities. This method has the potential to provide such insights; in addition it allows for the direct isolation of in situ mobilizing plasmids together with their endogenous hosts.

No MeSH data available.


Related in: MedlinePlus