Overexpression of csrA in L. biflexa.

(A) Overexpression of csrA in L. biflexa. To confirm overexpression of csrA, RNAs were extracted from each Leptospira strain and subjected to RT-qPCR. Results obtained from 3 independent cultures were presented as relative fold changes ± SEM. cysK was used for normalization. (***) indicated p-value <0.001. The growth curve of L. biflexa. The 2×10⁶ cells of each bacterial strain were grown in 10 mL of regular EMJH or 5-fold diluted EMJH in water, (B) represented growth in regular EMJH and (C) growth in 5-fold diluted EMJH. OD₄₂₀ measurement for growth was performed every 24 h. Results obtained from 3 independent experiments are expressed as Mean ± SEM. (D) Soft agar assay of L. biflexa. Leptospira OD₄₂₀ = 0.1 were inoculated onto 0.6% semisolid EMJH plate and incubated at 30°C. (E) Measurement of cell length of L. biflexa (F) Measurement of velocity of L. biflexa. Late exponential phase of Leptospira grown in EMJH medium were measured for cell length and velocity under a dark-field microscope using cellSens software (OLYMPUS).

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