Serotype-shifting gene rfbT is a direct transcriptional target of cAMP receptor protein (CRP) in V. cholerae O1

Serotype conversion gene rfbT is directly regulated by histone-like nucleoid structuring protein (H-NS) in V. cholerae O1

“…Though the gene rfbT has been recognized as the genetic determinant of Ogawa serotype of V. cholerae O1 serogroup for more than 30 years ( Stroeher et al, 1992 ) and various kinds of mutations were revealed in the rfbT coding sequence of isolates from different space–time sources ( Sharifnia et al, 2012 ; Liang et al, 2013 ; Karlsson et al, 2016 ), its regulation and the molecular structural features except the transcriptional start site, putative −35 and − 10 elements of the promoter-regulatory region remain unclear. Previously, we identified a cis -regulatory element, i.e., a non-canonical CBS in the promoter region, through which global regulator CRP exerts an activational effect ( Li et al, 2019 ). Further sequence analysis revealed that the G + C content of promoter-intergenic region of rfbT (41.2%) is quite low compared with the V. cholerae genome in general (47.7% for chromosome I and 46.9% for chromosome II).…”

“…The V. cholerae O1 El Tor biotype, Ogawa serotype strain C7258 was used as wild-type (WT) precursor (Peru isolate, 1991). The mutant Δ crp (WL7258) was generated previously ( Li et al, 2019 ). E. coli DH5αλ pir and S17-1λ pir were, respectively, used for cloning and conjugation purposes, ER2566 is used as host for the expression and purification of hns cloned into the pTXB1 vector.…”

“…The resulting pWM91-Δ hns or pWM91-Δ fis was introduced into C7258 or Δ crp (WL7258) by conjugation from S17-1λ pir . Exconjugants and mutants were selected as described previously ( Wu et al, 2015 ; Li et al, 2019 ).…”

“…Competing cold probe was amplified using the same primers without a biotin label. Probe-N7 ( Li et al, 2019 ) was a 140 bp fragment of rfbT promoter region containing the previously determined CRP binding site and used as a nonspecific control probe for H-NS binding ( Li et al, 2019 ). The reaction mixture of 15 ng biotin-labeled probe with increasing amounts of purified H-NS protein in reaction buffer (10 mM Hepes, 150 mM KCl, 1 mM EDTA, 1 mM DTT, 10 mM (NH 4 ) 2 SO 4 , 0.2% Tween 20) together with 100 ng BSA and 100 ng CF -DNA in each reaction (20 μl) was incubated at 28°C for 30 min and then separated on a 6% native polyacrylamide gel.…”

“…Such serotransitions are nonrandom processes and thought to be related to selective pressures of serotype-specific immunity within the host population or environmental stress, the specific intrinsic drivers and regulation mechanism remain to be investigated ( Longini et al, 2002 ; Karlsson et al, 2016 ). Previously, we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through directly binding to a non-canonical CRP binding site (CBS) in its promoter region ( Li et al, 2019 ).…”