Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4.

ABSTRACT

The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase high-performance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.