Fitness cost, gyrB mutation, and absence of phosphotransferase system fructose specific IIABC component in novobiocin-resistant Streptococcus iniae vaccine strain ISNO.

To understand the fitness cost of novobiocin-resistance in an attenuated Streptococcus iniae vaccine strain ISNO compared to its virulent parent strain ISET0901, cell proliferation rate of the two strains were compared to each other. Our results revealed that the cell proliferation rates of ISNO were significantly (P<0.05) smaller than that of ISET0901. To understand whether there was any mutation at the target site of novobiocin, DNA gyrase subunit B (gyrB) was sequenced from both strains. Sequencing results revealed a point mutation of AGA to AGC, resulting in a deduced amino acid substitution of R635S. To determine whether any unique DNA sequence was present in ISET0901 but absent in ISNO, PCR-select bacterial genome subtractive hybridization was performed. A phosphotransferase system fructose specific IIABC component sequence was confirmed to be present in ISET0901 but absent in ISNO. Using genomic DNAs from ten field-strains of S. iniae as templates, the phosphotransferase system fructose specific IIABC component sequence was found to be present in five highly virulent strains, but absent in five avirulent strains. Taken together, our results suggest that: (1) As fitness cost of novobicin resistance, ISNO had significantly smaller cell proliferation rate; (2) point mutation at target site gyrB resulting in R635S substitution was associated with novobiocin resistance in ISNO; and (3) phosphotransferase system fructose specific IIABC component was associated with virulence of S. iniae.