In-vivo validation

Overnight cultures of wild-type and ΔclpA Caulobacter crescentus strains each mixed at a 1:1 ratio with a reporter strain constitutively expressing fluorescent Venus (CPC798). The mixtures were kept at either 30°C or heat-shocked at 42°C for 45 minutes in a thermocycler. After the heat-shock, the mixtures were diluted to 1:4000 in PYE media and allowed to grow for 24 hours (∼12 doublings) at 30°C. Number of fluorescent control (Venus) and nonfluorescent tester (WT or ΔclpA) cells were counted in both the initial mixture and after 24 hour growth using phase contrast and fluorescent microscopy. The same tester and control normalization coefficients were used for initial and 24 hour time points for each strain (normalization coefficient = 1/(tester and control) at time = 0). and time = 24 by adjusting the time = 0 ratios to 1 for each strain. Normalized 24 hour ratios are what we are reporting as competitive index. An index of greater than one means the tester condition were able to grow faster compared to the control and an index of less than one means the tester grew slower compared to the control. Quantifications of at least 100 cells were performed for each condition with replicates when possible.