The WalR-WalK Signaling Pathway Modulates the Activities of both CwlO and LytE through Control of the Peptidoglycan Deacetylase PdaC in Bacillus subtilis

Abstract

The WalR-WalK two component signaling system in Bacillus subtilis functions in the homeostatic control of the peptidoglycan (PG) hydrolases LytE and CwlO that are required for cell growth. When the activities of these enzymes are low, WalR activates transcription of lytE and cwlO and represses transcription of iseA , a secreted inhibitor of LytE. Conversely, when PG hydrolases activity is too high, WalR-dependent expression of lytE and cwlO is reduced and iseA is de-repressed. In a screen for additional factors that regulate this signaling pathway, we discovered that over-expression of the membrane-anchored PG deacetylase PdaC increases WalR-dependent gene expression. We show that increased expression of PdaC, but not catalytic mutants, prevents cell wall cleavage by both LytE and CwlO, explaining the WalR activation. Importantly, the pdaC gene, like iseA , is repressed by active WalR. We propose that de-repression of pdaC when PG hydrolase activity is too high results in modification of the membrane-proximal layers of the PG, protecting the wall from excessive cleavage by the membrane-tethered CwlO. Thus, the WalR-WalK system homeostatically controls the levels and activities of both elongation-specific cell wall hydrolases. Importance: Bacterial growth and division requires a delicate balance between the synthesis and remodeling of the cell wall exoskeleton. How bacteria regulate the potentially autolytic enzymes that remodel the cell wall peptidoglycan remains incompletely understood. Here, we provide evidence that the broadly conserved WalR-WalK two-component signaling system homeostatically controls both the levels and activities of two cell wall hydrolases that are critical for cell growth.

Identification of genes required for swarming motility in Bacillus subtilis using transposon mutagenesis and high-throughput sequencing (TnSeq)

“…Transposon mutagenesis and high-throughput sequencing (TnSeq) requires a large library of mutants with diverse insertion sites. We chose a mariner-based transposon for library generation because it has been found to insert randomly at TA sequences, a pattern that is enriched in the low G+C content bacterium B. subtilis (29-31). Based on preliminary mutagenesis using standard conditions and plating, approximately 7% of colony forming units were transposants.…”

The WalRK two-component system is essential for proper cell envelope biogenesis in Clostridioides difficile

“…(As an aside, it is worth noting that repression of murE under Wal-ON conditions is not what one would expect if WalR promotes increased PG synthesis, as in B. subtilis (11,57)).…”

Two broadly conserved families of polyprenyl-phosphate transporters

“…Transposon libraries were generated as described previously ( 2 ), libraries were generated using pIR242, an E. coli - B. subtilis shuttle vector containing a temperature-sensitive replicon for B. subtilis , the mariner-Himar1 transposase, and a spectinomycin resistance cassette followed by a strong outward-facing promoter (P pen ) and flanked by inverted repeats recognized by the transposase. The pIR242 plasmid was separately transformed into and BIR769 or BIR672 and plated on LB agar supplemented with MLS and incubated at 30 °C.…”

“…Sequencing and mapping of the transposon insertion sites was performed as described previously ( 2, 3 ). Briefly, transposon-mutagenized colonies that grew on LB agar supplemented with MX2401 were pooled, and genomic DNA extracted.…”

“…We reasoned that over-expression of an UndP transporter would provide resistance to these antibiotics. We generated a Bacillus subtilis transposon library with a strong outward facing promoter ( 11 ) and plated the library on LB agar supplemented with MX2401 at concentrations above the minimum inhibitory concentration (MIC). The colonies were pooled and the transposon insertion sites mapped by transposon-sequencing (Tn-seq).…”