Pigmentation assay, stress responses, PHB quantification, iron deficiency, CAS assay and TPP riboswitch beta-lactamase assay

For quantifying pigment accumulation, 1 ml samples—obtained from a 5 day old broth culture grown at 37°C—were centrifuged at 16,000 × g for 10 min and supernatants measured at OD_550nm.

Total soluble protein was extracted by sonication and subsequent centrifugation. The protein amount of the supernatant was quantified by Bradford and 1μg of total protein was separated by SDS-PAGE, blotted on PVDF membranes and analyzed using anti-FlaA, CsrA and Flag antibodies as described before.

Sodium sensitivity was tested by plating serial dilutions of exponentially or post-exponentially grown bacteria on BYE agar containing or lacking 100 mM NaCl. CFUs were counted and percentage of sodium sensitive bacteria was calculated. For oxidative stress tests, L. pneumophila wt and csrA^- mutants were grown in BYE medium until exponential phase (OD2.5). Subsequently, the cells were pelleted and washed twice in PBS and resuspended in PBS at an optical density of OD 1.0. A final concentration of 5mM paraquat (Sigma-Aldrich) was added and the cultures were incubated for 2h at 37°C. At time point 0h, 0.5h, 1h and 2h samples were taken and serial dilutions were plated on BYCE plates to estimate bacterial survival. To analyze resistance to moderate acidic stress of wt and mutant strains, cultures were grown until OD 2.5, washed and diluted to OD 0.1 in BYE medium at pH ranging from 6.3 to 7.2. The bacteria were grown for 20h at 37°C under shaking and the OD was measured.