In a previous in silico study, we identified an essential outer membrane protein (LptD) as an attracti v e target for development of nov el antibiotics. Her e , w e c har acterized the effects of LptD depletion on Esc heric hia coli physiolo gy and morpholo gy. An E. coli CRISPR interference (CRISPRi) strain was constructed to allow control of lptD expression. Induction of the CRISPRi system led to ∼440-fold reduction of gene expression. Dose-dependent growth inhibition was observed, where strong knockdown effectively inhibited initial growth but partial knockdown exhibited maximum overall killing after 24 h. LptD depletion led to morphological changes where cells exhibited long, filamentous cell shapes and cytoplasmic accumulation of lipopolysacc haride (LPS). Tr anscriptional profiling by RN A-Seq show ed that LptD knoc kdown led to upre gulation of carbohydr ate meta bolism, especiall y in the colanic acid biosynthesis pathw ay. This pathw ay w as further ov er expr essed in the pr esence of sub lethal concentrations of colistin, an antibiotic targeting LPS, indicating a specific transcriptional response to this synergistic envelope damage. Additionally, exposure to colistin during LptD de pletion r esulted in downr egulation of pathw ays r elated to motility and c hemotaxis, tw o important virulence tr aits. Altogether, these results show that LptD depletion (i) affects E. coli survi v al, (ii) upr e gulates carbohydr ate metabolism, and (iii) synergizes with the antimicrobial activity of colistin.