Fig 2. The mlaA-knockout mutant exhibits various sensitivities to antibiotics and resistance to silkworm antimicrobial substances.

(A) E. coli overnight culture of the Parent/pMW118, mlaA::Tn/pMW118, or mlaA::Tn/pMW118-mlaA strain was 10-fold serially diluted; spotted onto LB agar plates supplemented with or without vancomycin, levofloxacin, chloramphenicol, tetracycline, CTAB, cholic acid, or chlorhexidine; and incubated at 37˚C. To examine the sensitivity to oxacillin and ampicillin, the mlaA deletion mutant (markerless deletion mutant of mlaA) transformed with pMW218 (ΔmlaA/pMW218) and the mlaA deletion mutant transformed with pMW218-mlaA (ΔmlaA/pMW218-mlaA) were used. (B) E. coli strains of the Parent/pMW118, mlaA::Tn/pMW118, or mlaA::Tn/pMW118-mlaA were aerobically cultured in LB medium and silkworm hemolymph was added to the bacterial culture at 40 min after the bacterial inoculation. The vertical axis represents the OD₆₀₀ value of the bacterial culture, and the horizontal axis represents the culture time. The means ± standard errors from 5 independent experiments are shown. Star indicates Student t-test p value less than 0.05 between the Parent/pMW118 vs. mlaA::Tn/pMW118, and between mlaA::Tn/pMW118 vs. mlaA::Tn/pMW118-mlaA. (C) E. coli strains of the Parent/pMW118, mlaA::Tn/pMW118, or mlaA::Tn/pMW118-mlaA were aerobically cultured in LB medium and the OD₆₀₀ values of the cultures were measured.