The regulation of competence for the DNA exchange in the biofilms of Bacillus subtilis

Bakterija Bacillus subtilis uravnava izražanje kompetence s sistemom zaznavanja kvoruma, ki temelji na kopičenju signala ComX, ki se veže na receptor ComP, kar sproži fosforilacijo in aktivacijo transkripcijskega faktorja ComA in s tem aktivacijo kompetenčnih genov v začetku stacionarne faze. Mutanta, ki ne producira signala (ΔQ), ni kompetentna za transformacijo, razen če ji dodamo ComX ali jo gojimo v kokulturi z divjim tipom, ki producira ComX. Osredotočili smo se na primerjavo frekvence transformacije in izmenjave DNA divjega tipa in dveh mutant (ΔQ), ki ne proizvajata signalnega peptida ComX. Naši rezultati so potrdili, da način priprave mutante vpliva na njeno občutljivost na signal, pri čemer se mutanta, ki ne proizvaja signala ComX, zaradi insercijske kasete za antibiotsko odpornost v genu comQ. Mutanta s kaseto se odziva močneje na ComX kot mutanta brez antibiotičnega označevalca (ML) se odziva na ComX podobno kot divji tip. Preverjali smo tudi ali način namnoževanja (statično / stresano) vpliva na frekvenco transformacije in na izmenjavo DNA. Ugotovili smo, da je transformacija višja v statični kulturi kot v stresaniizmenjava DNA med izogenimi sevi pa je višja v stresani kokulturi. Predpostavili smo tudi, da bo frekvenca izmenjave DNA višja v statični kulturi ne glede na sorodnost med sevi. To predpostavko smo zavrgli, saj je bila izmenjava DNA pri nesorodnih sevih (PS-216 : PS-196) višja v statičnem načinu namnoževanja kot v stresani kokulturi.The competence of Bacillus subtilis is regulated by the accumulation of the signal ComX. The signal binds to the receptor ComP and this triggers the phosphorylation and activation of the transcription factor ComA. ComA is responsible for the activation of competence genes in the early stationary phase. The mutant, which is not capable of signal production (ΔQ), is not competent unless we add ComX or it grows in coculture with the wild type ComX producer. By comparing the transformation frequency and DNA exchange frequency of the wild type and two mutants (ΔQ) that do not produce the signal ComX, we show that the approach used to construct a signaling mutant affects its sensitivity to the signal. Transformation frequency of the signal mutant is higher if the mutant has an antibiotic resistance cassette introduced in the comQ gene. The mutant without an antibiotic marker shows a similar response to ComX as the wild type. In addition we test how static versus planktonic growth influence transformation and DNA exchange frequency and whether the latter is influenced by kinship between strains. Results show that transformation frequency is higher in the static culture, while the DNA exchange frequency prevails in the shaken coculture. This is in contrast to DNA exchange frequency between two non-kin strains (PS-216 : PS-196), that exchanged DNA more efficiently when grown in static culture