Figure 1.
Megaplasmid Acquisition Decreases Thermal Tolerance.
A) A dilution series of strains DBL332/DBL365 (white) and DBL390/DBL412 (blue), which lack/contain megaplasmid pMPPla107 were plated at either 27°C or 37°C. P. stutzeri normally grows at both temperatures (left), but strains containing the megaplasmid (right) only grow at 27oC. Pictures were taken at the lowest dilution with growth (approximately 1∶1×109). White circle reflects where megaplasmid containing strains were spotted but didn't grow. B) Competitive fitness assays demonstrate that fitness of megaplasmid containing strains is significantly lower at 35°C compared to 27°C (p = 0.01). Fitness is normalized so that P. stutzeri lacking the megaplasmid is 1, error bars shown are +2 standard errors.
Figure 2.
Megaplasmid Acquisition Decreases Resistance to 7-hydroxycoumarin and Nalidixic acid.
A) A dose response curve comparing bacterial growth (OD600) of bacterial strains which contain (grey, DBL365) or lack (black, DBL332) the megaplasmid pMPPla107 across 7-hydroxycoumarin concentration. Curve is representative of at least 3 different assays. B) A dose response curve comparing bacterial growth (OD600) of bacterial strains which contain (grey, DBL365) or lack (black, DBL332) the megaplasmid pMPPla107 across nalidixic acid concentration. Curve is representative of at least 3 different assays. C) Competitive fitness assays demonstrate that fitness of megaplasmid containing strains is significantly lower in 4 µg/mL nalidixic acid compared to 0 µg/mL (p = 0.046). Fitness is normalized so that P. stutzeri lacking the megaplasmid is 1, error bars show +2 standard errors in all cases.
Figure 3.
Megaplasmid Acquisition Decreases Biofilm Formation.
A) Four day old cultures of strains which lack (DBL332, right) or contain (DBL365, left) megaplasmid pMPPla107 grown in M9 media supplemented with 10 Mm succinate. Total population sizes in both cultures are roughly equal, but turbidity differences arise because almost all cells without the megaplasmid are found within the biofilm mass. B) Pipette tips harvested after four days of growth in 2 mL SWLB media cultures for either DBL365 (left) and DBL332 (right). C) Bacterial population sizes of DBL332 (grey) and DBL365 (white) after four days of growth in 21mL cultures of SWLB media. Population sizes of planktonic bacteria are shown on the left, while those harvested from pipette tips are shown on the right. Error bars show +2 standard errors in all cases.
Figure 4.
Megaplasmid Acquisition Increases Motility or Chemotaxis.
A) Halo size in soft agar is significantly larger for bacterial strains which contain the megaplasmid (DBL365, black) compared to those that lack it (DBL332, grey). Error bars show +2 standard errors. B) Average difference in halo size represented in circular form. Grey circle is P. stutzeri while black circle represents P. stutzeri containing the megaplasmid.
Figure 5.
Megaplasmid Acquisition Increases Sensitivity to P. aeruginosa Supernatant.
Purified supernatant from P. aeruginosa PA14 was spotted on lawns of P. stutzeri that either lacked (DBL332, left) or contained (DBL365, right) megaplasmid pMPPla107. White circle on left panel represents where supernatant was spotted onto lawn where P.stutzeri continued to grow. Right panel shows clearing area in lawns where supernatant was spotted. Photo is representative of >3 independent experiments.
Table 1.
Strains and Plasmids.