Role of RfaH in Yersinia enterocolitica O:3

To assess the effects of rfaH mutation on gene expression, RNA sequencing was carried out using the RNA extracted from rfaH mutant and wild type bacteria cultivated at both 22°C and 37°C. Additionally, to differentiate between genes affected directly by the rfaH mutation and those affected by the O-antigen negative phenotype, the Y. enterocolitica O:3 strain YeO3-R1 missing the O-antigen was included in the RNA-sequencing study. At RT-grown bacteria altogether 77 (45 up- and 32 down-regulated) differentially expressed genes were identified. At 37ºC, on the other hand, 44 genes were differentially expressed; 14 genes up- and 30 genes down-regulated. The RNA-sequencing data of YeO3-R1 was available only for bacteria grown at 22°C and the comparison revealed that 22 of the 102 genes were similarly differentially expressed both in rfaH and YeO3-R1 mutants. To detect genes regulated directly or indirectly by Hfq, deep RNA-seq was performed with RNA isolated from Y. enterocolitica wild-type strain 6471/76 and the YeO3-hfq::Km strain. Total RNA was isolated from two biological replicas of bacteria grown in LB to logarithmic phase (OD600 = 0.6) at two different temperatures, RT and 37°C. knocking out of Hfq affected the transcription of 346 genes at RT and 541 genes at 37°C, i.e., ca. 8 and 12.5 % of the Y. enterocolitica genes, respectively. At RT, of the 346 genes 216 genes were down- and 132 up-regulated, and at 37°C, of the 541 genes, 96 were down- and 445 up-regulated. A total of 95 genes were differentially expressed both at RT and 37°C; 27 showed down- and 59 up-regulated. For 9 genes opposite changes took place depending on the growth temperature. Overall design: Bacterial RNA profiles of wild type, rfaH- and hfq- strains were generated using deep sequencing, in duplicates, under two growth temperatures (22°C and 37°C). The RNA-sequencing data of YeO3-R1 was generated only from bacteria grown at 22°C.