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None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. aimed to investigate whether deletion of various peptidoglycan hydrolase genes had any effect on competence development in B. subtilis. The bacterial strains and plasmids used in this study are listed in Table 1. The oligonucleotide primers are listed in Table 2. Escherichia coli DH5a was used as a host for cloning purposes. To determine the transformation efficiency, various B. subtilis cells were first grown at 37∞C in the absence of any antibiotic as described by Contente and Dubnau (1979) for the preparation of competent cells. The period of starvation for competence development was usually 60 min unless otherwise specified. One milliliter of competent cells were then incubated at 37∞C with the shuttle plasmid pHY300PLK (final concentration: 3.7 ng ml–1) for 1 h, and were subjected to serial dilution and plating on Luria-Bertani (LB) agar plates (containing 10 g l–1 tryptone, 5 g l–1 yeast extract, 5 g l–1 NaCl, and 15 g l–1 agar) supplemented with or without tetracycline (10 mg ml–1). Transformation efficiency was expressed as the percentage of Tcr transformants of the total viable count. The results showed that deletion of the peptidoglycan hydrolase gene cwlO or lytE severely impaired plasmid DNA transformation efficiency (Table 3). In contrast, deletion of cwlS, lytC, lytD, or lytF had no apparent effect on competence development (Table 3). To perform complementation experiments, we constructed the pDL (Yuan and Wong, 1995)-based integrative plasmid pGS2749 (Table 1), which carries an intact cwlO gene expressed from its native promoter (amplified by PCR with the primer pair B419 plus B420), and utilized the pDL-based integrative plasmid pENB14 (Tseng et al., 2011), which carries an intact lytE gene expressed from its native promoter. These two plasmid constructs were then individually introduced into competent cells of Deletion of the cell wall peptidoglycan hydrolase gene cwlO or lytE severely impairs transformation efficiency in Bacillus subtilis

journal of general and applied microbiology

Deletion of the cell wall peptidoglycan hydrolase gene &lt;i&gt;cwlO&lt;/i&gt; or &lt;i&gt;lytE&lt;/i&gt; severely impairs transformation efficiency in &lt;i&gt;Bacillus subtilis&lt;/i&gt;