Silencing of essential genes within a highly coordinated operon in XXXX .

@entity23782 @entity851 @entity526 @entity14241
UNASSIGNED: Operon-encoded essential bacterial genes are particularly challenging to study independently because of coordinated gene expression and the non-viability of knock-out mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene-disruption of genes may help establish essentiality, but can lead to polar effects on genes downstream or upstream of the target gene. Here, the @entity526 ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. Gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition, as a minimum transcript level 50 (MTL50) value. @entity23782 and @entity14241 , the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and ORF-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL50 mean and SD values of ribF, ileS, and lspA were 0.65    0.18, 0.64    0.06, and 0.76    0.10, respectively. However, they were not significantly different (p = 0.71, weighted one-way ANOVA). Gene requirement for ispH could not be determined due to @entity851 . @entity23782 and @entity14241 sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA silencing antimicrobials.
