2.1. Creation of yycH, yycI, and yycHI Antisense Knockdown Clones and yycHI Deletion

Primers AS-yycH_for, AS-yycH_rev, AS-yycI_for, AS-yycI_rev, AS-yycHI_for, and AS-yycHI_rev (Table 1) were used to create antisense inserts for the yycH (223 bp) and yycI (189 bp) genes as well as a region overlapping the last 67 bp of yycH and the first 100 bp of yycI for the inhibition of transcription of both genes. The PCR products were digested with XbaI and EcoRI and ligated into the pEPSA5 vector [25] in antisense direction, resulting in the antisense plasmids pEPSA5-AS-yycH, pEPSA5-AS-yycI, and pEPSA5-AS-yycHI. The constructs were first transformed into CaCl₂-competent Escherichia coli JM109 cells, then electroporated into S. aureus RN4220 and finally into S. aureus HG003 cells, which is a derivative of the widely used model strain S. aureus NCTC 8325, in which the mutations in tcaR and rsbU have been repaired [26]. Sanger sequencing confirmed all constructs. Reduction of the protein expression was controlled by Western blotting with specific anti-YycH and anti-YycI antibodies (Figure 2). The pIMAY-ZΔyycHI deletion vector was constructed as described previously [9]. The plasmid was transformed into S. aureus NRS384 walR-FLAG with allelic exchange conducted as described by Monk et al. [27]. S. aureus NRS384 is a member of the USA300 community associated epidemic MRSA [9].

Characterization of S. aureus HG003 with antisense knockdown of the YycH and YycI proteins. (a) Western blots of S. aureus HG003 and S. aureus NRS384 walR-FLAG lysates with the pEPSA5, pEPSA5-AS-yycH, pEPSA5-AS-yycI, and pEPSA5-AS-yycHI vectors as well as recombinant YycH-His6 and YycI-His6. Antisense RNA against yycHI reduced the translation of both proteins. Knockdown of yycH reduced the abundance of YycI and vice versa. Expression of the same antisense plasmids in S. aureus NRS384 walR-FLAG showed no significant reduction of WalR abundance. (b) Triton X-100 induced autolysis is reduced in the S. aureus HG003 yycHI knockdown strain. Cells of S. aureus strains HG003 pEPSA5 and HG003 pEPSA5-AS-yycHI were harvested in late-log phase, washed, and resuspended in TBS containing 0.1% Triton X-100. Lysis was observed by OD₆₀₀ measurements every 30 min. Error bars indicate standard deviation of two independent experiments. Similar results were obtained with S. aureus RN4220 pEPSA5 and RN4220 pEPSA5-AS-yycHI (data not shown). (c) Zymogram of LiCl autolysin extracts from S. aureus strains NRS384 walR-FLAG (1), NRS384 walR-FLAG ΔyycHI (2), HG003 pEPSA5 (3), HG003 pEPSA5-AS-yycHI (4). (5): PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific, Schwerte, Germany). Pro-AtlA (138 kDa): Full-length AtlA with propeptide; AtlA (113 kDa): Full-length AtlA without propeptide; PP-AM (87 kDa): Amidase domain with propeptide; AM (62 kDa): Amidase domain; GL (51 kDa): Glucosaminidase domain; Designations according to [28]. For better visibility, the image of the methylene blue stained gel was converted to greyscale and inverted. The antisense knockdown of yycHI in HG003 as well as deletion of the yycHI genes in NRS384 resulted in decreased lysis of M. luteus in the zymogram; (d) Inhibition of yycHI transcription increases WTA content of the cell wall. Cell walls of S. aureus strains HG003 pEPSA5 and HG003 pEPSA5-AS-yycHI were purified and WTA content was compared by determination of the inorganic phosphate content. Error bars indicate standard deviation of three independent experiments. p value of two-tailed t-test was 0.0149 and considered significantly different (p < 0.05). (e) UPLC analysis of mutanolysin-digested peptidoglycan of S. aureus HG003 pEPSA5 and HG003 pEPSA5-AS-yycHI. Induction of the yycHI antisense knockdown did not cause changes in muropeptide composition and cross-linking.

Primers used in this study.