3 PROYECTOS DE RESPONSABILIDAD SOCIAL EN EL FONDO
3.5 PROYECTOS FOEMETCOL AÑO 2010
the same gene (carB).
3. To test whether ecological interactions have an impact on the likelihood of switcher evolution in genotypes from different evolutionary time points. Here, variation in the number of re-‐evolved switchers should be linked to a change in population composition (i.e. number of new variants, cell density, diversity) over evolutionary time.
5.2
Summary of materials and methods
5.2.1 Experimental design of the SREE in Line 1
Experimental conditions of the SREE were based on the experimental design of the original REE and followed the same experimental guidelines (see section 1.4.4). In summary, during the REE bacterial cultures were grown for three days in one of the selective environments, static or shaken, which marked one selection round. After three days the bacterial cultures were plated and screened for a new colony morphology type. The most abundant new type was selected for the inoculation of a new microcosm, which was then kept under the opposite environmental condition than that in which it had evolved (the next selection round). This was repeated for several selection rounds, with an alternation between a static and a shaken environment for each selection round. In cases when no new type was detected, a small amount of culture was transferred into a new microcosm and kept for another three days under the same environmental condition (first transfer, same selection round). This was repeated until a new type was detected on agar plates (several transfers in one selection round). The appearance of a type with novel colony morphology marked the end of a selection round.
In this study a replay experiment (SREE) was performed from different starting positions (evolutionary time points) in Line 1. The SREE was carried out to test whether the evolution of a switcher genotype is equally likely from any genotype
that evolved at different time points during the course of evolution or whether the eight preceding mutations that were present in 1s4 (immediate ancestor of the original switcher in the REE) are essential. The switcher in the REE occurred in Line 1 and Line 6 after eight rounds of selection (see section 1.5). The focus of this study is on Line 1 as it has been characterised and studied to a great extent in previous studies (Beaumont et al., 2009; Gallie, 2009). The switcher in Line 1 (1w4) evolved in a static environment from 1s4.
Table 5.1: Mutational history of the switcher (1w4) in Line 1. Evolutionary history of the switcher in Line 1 is composed of nine mutations, including the switcher mutation
carB* (last column). Mutations that occurred during history occurred in genes that are involved in the regulation of cellulose synthesis due to selection in a static or in a shaken environment. The founder populations were genotypes that would, according to the experimental design of the REE, go into a static environment in the following selection round (“s” genotypes; bold) because the original switcher (1w4) evolved under static conditions.
Number of
mutations 1 2 3 4 5 6 7 8 9
Environment static shaken static shaken static shaken static shaken static
Name 1w0 1s1 1w1 1s2 1w2 1s3 1w3 1s4 1w4
Mutational
History mwsR mwsR awsX awsR wspF wssA mwsR mwsR carB*
For that reason five genotypes that occurred during the REE in Line 1 and would grow according to the rules of the REE in the next selection round in a static environment were selected (Tab. 5.1; SBW25, 1s1, 1s2, 1s3, and the immediate ancestor 1s4). The strains were revived on KB agar plates from a frozen stock culture.
The experiment was divided into three experimental blocks with 20 replicates of each strain in each block (60 replicate microcosms in total for each lineage). For one biological replicate a single colony was picked from the agar plate and transferred into 6 ml liquid KB media. After growing overnight, 6 µl of each culture
for three days (one selection round). After three days the cultures were thoroughly vortexed, diluted and spread on KB agar plates (Fig. 5.2).
Figure 5.2: Experimental design of the SREE. Overnight cultures of SBW25, 1s1, 1s2, 1s3 and 1s4 acted as founder populations to replay the evolution of phenotypic switching under static conditions (60 microcosms per lineage). After three days’ growth in a static microcosm the cultures were diluted, plated and screened for new colony types. If a new type or a switcher (new/SW) was detected the replicate was terminated. Switchers were stored at –80°C. If no new type was detected 6 µl of the culture were transferred to a new microcosm and incubated for another three days under static conditions before being diluted, plated and screened for new types again. This process was repeated until a new type was found.
The OD600 was randomly measured for two to three microcosms in each strain to
estimate the plating volume that was needed for four plates to be able to screen approximately 500-‐1000 colonies. After two days all plates were screened for new colony types. In addition one plate from each replicate was randomly chosen to count the numbers of ancestral colonies, the numbers of the most common colony type, the total numbers of new type colonies, numbers of switcher colonies and colony diversity (number of different colony morphologies). As soon as a new
Founder( popula,ons( SBW25& 1s1& 1s2& 1s3& 1s4& 1.&&
Transfer& Transfer&2.&& Transfer&3.&&
6(µl(( ((
6(µl((
(( 6(((µl((
.&.&.&&
new/SW( new/SW( new/SW( new/SW(
switcher&stored&at&980°C( & & & un,l(new( type(was( found( (
colony type was found on one of the four plates, the replicate line was terminated (Fig. 5.2).
Colonies that showed switching characteristics were transferred to new KB agar plates from which a single colony was used to inoculate an overnight culture, which was then stored at -‐80°C the following day. In addition, the new switching
type was transferred to new agar plates several times in a row to certify the inheritance of the novel phenotype. Furthermore, negative staining with Indian ink confirmed a switcher phenotype on the cellular level under the microscope (see Chapter 1, Fig. 1.9C). Where no new type was observed on the plates, 6 µl of the
three-‐day culture were transferred to a fresh microcosm, which was left undisturbed for another three days under the same environmental conditions (first transfer). This procedure was repeated parallel to diluting, plating and screening until a new type was found (several transfers; Fig. 5.2).
5.2.2 Screening the carB gene of re-‐evolved switchers
The switcher genotypes that evolved during the course of the SREE were further analysed and screened for an underlying mutation in the carB gene. A PCR was performed with the primer pair SN039 PF and SN040 PR, using the protocol that was described earlier (see Chapter 2, section 2.2.1.1). The PCR products were sequenced using carB specific primers (SN009 PF, SN010 PF, SN013 PF, SN014 PF, SN015 PF, SN016 PF, SN017 PF, SN018 PF and SN070 PR; see Chapter 2, Tab. 2.3) and screened for changes in the DNA sequence by comparison to the P. fluorescens SBW25 ancestral DNA sequence using the alignment software Sequencher.
5.2.3 Statistical analyses
The software JMP 9.0 was used to perform generalised linear models using the data obtained during the SREE. The data included number of switcher that were
detected on plates after the dilution of a population containing 109 cells as well as
number of new colony types and number of ancestors. Illustrations of the results were created with the software Prism 5.
5.3
Results
5.3.1 The re-‐evolution of switching genotypes in SBW25, 1s1, 1s2, 1s3 and 1s4 (SREE)
5.3.1.1 Total number of re-‐evolved switcher genotypes
The founder lineages SBW25, 1s1, 1s2, 1s3 and 1s4 evolved in Line 1 during the course of the REE at different evolutionary time points (see Chapter 1, section 1.4.4). The evolution of a switcher genotype from 1s4 in a static environment was already observed during the course of the REE (Beaumont et al., 2009, Gallie, 2009). In this study I was interested in the likelihood of switcher occurrence at earlier time points represented by the original common ancestor SBW25 and the genotypes 1s1, 1s2 and 1s3 in addition to the immediate ancestor of the switcher, 1s4. The average number in 60 replicate microcosms in three experimental blocks (N = 3) that produced a switcher genotype was then calculated. The results of a generalised linear model show that the different genotypes vary in their probability to produce switcher genotypes (X26 = 17.96, P < 0.01). Most of the
difference was explained by the lineage itself (X24 = 43.16, P < 0.0001). The
genotypes 1s3 and 1s4 had the highest number of microcosms that produced a switcher (Fig. 5.3) and showed a similar likelihood of switcher occurrence (X24 =
2.9 10-‐31, P = 1). Both were distinctive from the other lineages in their ability to
produce switchers (X24 = 16.47, P < 0.0001). No switcher was detected in 1s1 (Fig.