Since deletion of JAK inhibitor SA1665 has been shown to increase β-lactam resistance, reduced SA1665 transcription in the presence of β-lactams may also provide some protection against β-lactam exposure.
Figure 5 Northern and Western blot analyses. A, Transcription of SA1665 over growth in CHE482, with RNA harvested at the OD600 nm values indicated. B, Transcription of SA1665 from CHE482 grown to OD600 nm 0.25 and either left uninduced Trichostatin A (-) or induced with either 4 or 120 μg/ml of cefoxitin fo 0′, 10′ and 30′. C, Transcriptional profiles of SA1664, SA1665, SA1666 and SA1667 in CHE482 and ΔCHE482, grown to OD600 nm 0.25 and either uninduced or induced with cefoxitin 4 μg/ml for 0′, 10′ or 30′. Approximate sizes of transcripts, in kb, are indicated on the right of the blots. D, Transcription of mecA and mecR1 in CHE482 and ΔCHE482, grown to OD 0.25 and either left uninduced or induced with cefoxitin (4 μg/ml) and sampled after 0′, 10′ and 30′. Ethidium bromide stained 16S rRNA bands from all Northern gels are shown as a comparative indication of RNA loading. E, Western blots showing amounts of PBP2a in ZH44 and ZH73 Lazertinib solubility dmso and their respective
SA1665 deletion mutants, before (0′) and after induction with 4 μg/ml of cefoxitin for 10′ and 30′. Northerns also showed that, as expected, the SA1665 transcripts were absent from the deletion mutant (Figure 5C), and additional experiments demonstrated that wild type SA1665 transcription patterns were restored by complementation of ΔCHE482 with pME26 (data not shown). The effects of SA1665 deletion on directly up- and down-stream genes GBA3 were also investigated. Northern blots of the neighbouring genes SA1664, SA1666 and SA1667, showed that expression of all three genes was very weak compared to that of SA1665. A weak transcript of about 3 kb was present in hybridizations probed with orfs SA1665-SA1667. This band decreased in size in the SA1665 mutant when probed with SA1666 and SA1667. One of the transcripts hybridising
to the SA1664 probe also decreased in size by ~0.5 kb in the SA1665 mutant, suggesting that SA1665 was present on several transcripts of different lengths, including a high abundance monocistronic transcript and low abundance polycistronic transcripts (Figure 5C). Transcript abundance of both the upstream SA1666-SA1667 operon and the downstream SA1664-specific transcript all appeared to increase slightly in ΔCHE482. The significance of these subtle increases in transcription are unknown, however, polar effects from SA1665 deletion seem unlikely, based on the facts that all genes were still transcribed, their transcription levels all remained extremely low and the transcriptional terminator of SA1665 remained intact in the deletion mutant (Figure 1B). Expression of mecR1 and mecA were analysed from RNA of uninduced and induced cultures of CHE482 and ΔCHE482. Cells were induced at OD600 nm 0.25 (Figure 5D) and 1.