No abstract

The peptidoglycan cell wall is a common target for antibiotic therapy but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated derivative of D39 Streptococcus pneumoniae. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analog, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins.

Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for σD-dependent gene expression: a pattern suggestive of bistability. The gene encoding σD, sigD, is part of the 31-gene fla/che operon where its location at the 3′ end, 25 kb away from the strong Pfla/che promoter, determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the chromosome inhibited σD-dependent gene expression. SlrA together with SinR and SlrR reduced sigD transcript by potentiating a distance-dependent decrease in fla/che operon transcript abundance that was not mediated by changes in expression from the Pfla/che promoter. Consistent with acting upstream of σD, SlrA/SinR/SlrR could be bypassed by artificial ectopic expression of sigD that hysteretically maintained for 20 generations by engaging the sigD gene at the native locus. SlrA/SinR/SlrR was also bypassed by increasing fla/che transcription and resulted a hypersensitive output in flagellin expression. Thus, flagellin gene expression demonstrated hypersensitivity and hysteresis and we conclude that σD-dependent gene expression is bistable.

No abstract

In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP.