Innova 44

For pre-inoculum preparation, a loopful of B. thuringiensis grown on LB plate was used to inoculate 3 ml of sterilized LB medium and incubated in a rotary shaker (New Brunswick Scientific, model INNOVA^® 44, USA) at 30 °C and 200 rates per minute overnight (14–18 h). For inoculum preparation, 250-ml Erlenmeyer flasks containing 50 ml of LB medium were inoculated with 1 % (v/v) of the pre-inoculum and incubated in a rotary shaker at 30 °C and 200 rates per minute for 6 h. The volume of culture inoculum was determined on the basis of a final absorbance of approximately 0.15 measured at 600 nm. The optical density at 600 nm (OD₆₀₀) was determined using a SmartSpec™ 3000 UV–visible spectrophotometer (Bio-Rad Laboratories). The 500-ml flasks containing 50 ml of complex medium were incubated with estimated inoculum volume. In such media, the initial OD was not measured after inoculation but calculated according to the OD measured in the inoculum. Samples taken periodically from the incubated cultures were subjected to microscopical examination. When 90 % (or more) of the B. thuringiensis cells had lysed, releasing the spores and crystals, the fermentation process was considered as finished.

Rhodothermus marinus (strain R-10 DSM 4252) was grown in ATCC Medium 1599 enhanced with NaCl (1% w/v) at 60–80 °C in an Innova44 (New Brunswick Scientific) shaking incubator either in unbaffled Pyrex^® Erlenmeyer flasks (2 L, filled to 0.6 L), or Tunair™ polypropylene flasks (2.5 L, filled to 1 L), shaken at 180 rpm.
When cultures reached an OD₆₀₀ 0.9–1.0, the cells were harvested by centrifugation and stored at − 80 °C. The cell paste was lysed using glass beads in a PreCellys homogenizer (sonication was found to be insufficient for effective cell lysis). Ribosomal proteins were purified using a previously reported method (Hardy et al. 1969 (link)).

Each population sample from the glycerol stock was used for inoculation of 25 ml medium and the culture got cultivated at 32 °C at 150 r.p.m for 72 h (New Brunswick Innova 44). After incubation, 1 ml culture was centrifuged at 15.000 × g for 1 min. Both the pellet and the supernatant were further treated according to a modified method of Gaitonde to determine the L-cysteine yields (Additional file 1: Note 1) [54 (link)].

The simulated long-term cultivation method was adapted from Rugbjerg et al. [21 (link)]. Single colonies of freshly transformed cells were inoculated into 250 ml baffled shaking flasks containing 25 ml medium. W3110 strains were cultivated at 32 °C for 10 h and horizontal shaking at 150 r.p.m (New Brunswick Innova 44). Each strain harbouring one out of three plasmids got cultivated in triplicates. Due to a higher growth rate of the MDS42 strain, cultures were kept at 32 °C just for 5 h to always maintain an exponential growth phase. After each time point cultures were inoculated into 25 ml fresh medium (starting OD600 = 0.05) and incubated under the same conditions for another 10 h or 5 h. At each passage, sample’s OD₆₀₀ were recorded to determine the accumulated generations (Additional file 1: Table S2) and 1 ml got snap-frozen with 1 ml 50% glycerol in liquid N2 and stored at − 80 °C. Subsequently the sampled cryo-cultures were re-cultivated for 72 h with a starting OD₆₀₀ of 0.01 into 25 ml fresh medium under above mentioned conditions. Growth rates were monitored every 30 min. L-cysteine yield determination was performed after 72 h. RNA extraction was carried out at OD₆₀₀ values of 0.6–0.8. Extraction of plasmid DNA was performed from the same culture (Fig. 2).

A methicillin-resistant S. aureus LAC strain expressing GFP (AH1726) was used for all confocal microscopy, image analysis quantification, and growth curves. For macroscopic imaging experiments, the quadruple mutant AH4413 (ΔclfA ΔfnbAB clfB::Tn) was utilized along with the wild-type strain, AH1263 (35 (link)). Cultures were streaked on tryptic soy agar (TSA) (BD Biosciences, Heidelberg, Germany) from agar slants (Horswill Laboratory, University of Colorado School of Medicine, Aurora, CO). TSA plates were incubated overnight at 37°C, and a single isolated colony was used to inoculate 5 mL of tryptic soy broth (TSB) (BD Biosciences) in a 15-mL Falcon tube. Inoculated broth cultures were grown for 18 h at 37°C in an orbital shaker set to 200 rpm (Innova 44; New Brunswick Scientific).

Neutrophil suspensions with different neutrophil concentrations ranging between 1 × 10⁶ and 15 × 10⁶ neutrophils/ml were prepared in Hepes buffer in the presence or absence of 40% human serum. Bacteria were added to the neutrophil suspensions with different multiplicities of infection (MOI) ranging from 1 to 10. After addition of the bacteria the suspension was mixed gently and put in an incubator (Innova^® 44; New Brunswick Scientific, Edison, NJ, USA) at 37°C and 180 rpm. One sample of neutrophil–bacteria suspension was kept on ice to serve as a negative control. At different time points samples were taken from the neutrophil–bacteria suspension and fixed with 1% paraformaldehyde (PFA) for ≥15 min on ice.