It is surprising, therefore, that both microarray and qRT-PCR data showed that the capacity of the EC-ECg combination to modulate EMRSA-16 gene expression was reduced compared to ECg alone; this is likely to indicate that the position adopted within the bilayer by ECg, in the absence of EC, maximizes the strength of the signals governing reconfiguration of cytoplasmic membrane architecture. Thus, although vraX was strongly up-regulated by the catechin combination as judged by qRT-PCR, it was significantly less than that found with ECg alone. Similarly, transcription of dltA, mecA, fmtA and isaA were significantly upregulated by ECg but modulation of these genes failed to reach levels of statistical significance when cells were exposed simultaneously to ECg and EC. Differences in the capacity of ECg and EGCg to interact with lipid bilayers suggested that changes to the B-ring hydroxylation pattern could influence the bioactivity of galloyl catechins. It was therefore surprising that 2 failed to induce ‘‘over-compensation’’ of membrane fluidity after 4 h incubation in a similar way to ECg and compounds 1 and 3; there appears to be no obvious reason for this highly reproducible finding as in other respects ECg, 1, 2 and 3 represent a series of compounds with progressively enhanced membrane-perturbing properties. Although we have previously found little or no evidence, it remains a possibility that the profound effect of 2 on virulence gene expression and relatively low impact on membrane fluidity is due to interactions with elements of the stress regulon or other two-component systems; this will be investigated in future studies. Compound 3, carrying no hydroxyls on the Bring, was associated with an increased oxacillin susceptibilitymodifying capacity, and induced both a highly fluid staphylococcal cytoplasmic membrane comparable to the ECg/EC combination and a level of gene modulation higher than other molecules in this series,JNJ-42153605 supporting the contention that progressive removal of –OH groups from the B-ring increased membrane interactions and bioactivity. In this context, 3 had a greater impact on the biophysical properties of LPG:PG:CL vesicles than ECg, strongly increasing the lipid order of fluid bilayers from a location close to the lipid/water interface. Compounds 4 and 5, in which all hydroxyl groups had been excluded from the A- and B-rings, possessed clear antibacterial activity and were poor resistance modifiers compared to ECg and analogs 1–3. Compound 5 impacted primarily on EMRSA-16 by disrupting the integrity of the cytoplasmic membrane, reflected in the induction of a different transcriptomic profile compared to molecules 1–3. The large majority of the genes selected for qRTPCR analysis LJI308 were significantly down-regulated at concentrations below the MIC, reflecting the overt antibacterial properties of this analog. Compound 5 dramatically altered the thermal characteristics of MLVs modelled on the S. aureus cytoplasmic membrane, shifting Tm to a lower temperature and creating a second peak of transition; 5 also appeared to render cells non-viable from a more superficial location in the membrane compared to ECg.This study has shown that progressive removal of hydroxyl groups from ECg effects a transition from molecules eliciting an extremely weak antibacterial effect but characterized by a capacity to induce a b-lactam-susceptible phenotype to overtly antibacterial compounds with limited b-lactam-resistance modifying properties.