Dataset Information

The Serine/Threonine kinase PrkC of Clostridioides difficile regulates export of CD1135, a septal peptidoglycan hydrolase

ABSTRACT: Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The activity of several enzymes involved in PG synthesis is regulated by serine/threonine kinase (STKs). In firmicutes, inactivation of genes encoding STKs is associated with a range of phenotypes including cell division defects and changes in cell wall metabolism, but only a few kinase substrates have been identified. We previously demonstrated that the STK PrkC plays an important role in cell division, cell wall metabolism and drug resistance in Clostridioides difficile. In this work, we identified and characterized a PGH, CD1135 that is a PrkC substrate. We showed that CD1135 hydrolyses PG between daughter cells to allow cell separation. We demonstrated that CD1135 phosphorylation inhibits CD1135 its export, therefore controlling the hydrolytic activity in the cell wall. High level of CD1135 in the cell wall leads to cell elongation, whereas low level causes cell separation defects. We provided evidences that the STK signaling pathway regulates PGHs homeostasis to control PG hydrolysis during growth and cell division.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Peptoclostridium Difficile (strain 630) (clostridium Difficile)

TISSUE(S): Culture Condition:anaerobically-grown Cell, Prokaryotic Cell

SUBMITTER: Thibaut Douché

LAB HEAD: Isabelle Martin-Verstraete

PROVIDER: PXD021541 | Pride | 2021-04-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190826_Q2_TD_ColQ2-63_DifKin_PhosTag_CD1135-CD2148.raw	Raw
	20190826_Q2_TD_ColQ2-63_DifKin_PhosTag_CD1135-prkC.raw	Raw
	20190826_Q2_TD_ColQ2-63_DifKin_PhosTag_CD1135.raw	Raw
	CD630_prot.fasta	Fasta
	txt.zip	Other

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Project description:Streptococcus pneumoniae is a human commensal bacterium that causes serious diseases. Peptidoglycan (PG) is the critical component of bacteria that maintains cell shape, size, chaining, and turgor resistance. Because of its surface exposure and eubacterial-specific mechanism of biosynthesis, PG biosynthesis remains an outstanding target for discovery of new antibiotics to resistant “superbugs,” like S. pneumoniae. Pneumococcal PG biosynthesis is carried out by a balance of septal and peripheral (side-wall-like) PG synthesis mediated by bPBP2x and bPBP2b, respectively, that emanates from midcell regions. We selected for additional suppressor mutations, besides mltG, that eliminate the requirement for essential bPBP2b in peripheral PG synthesis. We found that mutations that inactivate a putative RNA binding protein, designated KhpA, suppressed the requirement for some, but not all of the essential proteins required for peripheral PG synthesis. KhpA was used as bait in co-IP experiments to demonstrate interactions with a second RNA binding protein, designated KhpB, in cells. Mutations in khpA or khpB phenocopy each other and result in slower growth rates, smaller cell size with normal aspect ratios, and induction of cell wall stress responses. RIP-Seq analyses confirmed KhpAB as RNA binding protein in cells. Another suppressor of Δpbp2b implicated induction of a cell division operon as a way to bypass the requirement for bPBP2b. We show that increased expression of this cell division operon occurs in ΔkhpAB mutants and that this induction is necessary and sufficient to account for suppression of Δpbp2b. Comparisons of relative transcript amounts and protein levels indicate that induction of this cell division operon in ΔkhpAB mutants occurs primarily at the post-transcriptional level. Together, our results show that KhpAB, which are virulence factors in S. pneumoniae and are conserved in other Gram-positive pathogens, acts as a new class of RNA binding protein, with properties analogous to Hfq in Gram-negative bacteria.

Project description:Background: Mtb's cell wall comprises peptidoglycan, arabinogalactan, and mycolic acids linked to capsule proteins and polysaccharides by noncovalent bonds. Cell division requires extensive remodeling by inserting cell wall-building subunits, which require multiple enzymes to ensure precision and accuracy during the addition and conjugation of biomolecules to the cell wall. Approximately 35% of division and cell wall cluster operon genes are involved in cell wall biosynthesis, 22% in cell division, and 43% are still unstudied. Results: Rv2166c was examined in M. smegmatis, a substitute for M. tuberculosis, using three strains: CRISPR-Cas12a-knockout ∆Ms_4236, complemented ∆Ms_4236::Rv2166c, and pALACE transformed Ms_Vec were grown at 370C in 7H9 or 7H10 to evaluate phenotypic, chemical stress, and antibiotic responses in normoxia and hypoxia. We also examined the whole transcriptome to identify genes associated with MSMEG_4236 deletion of the Rv2166c homolog under normoxia and hypoxia. Deletion and overexpression of Rv2166c affect mycobacterial biofilm formation, cell elongation, colony morphology, and sliding motility in normoxia but are alleviated in hypoxia. Overexpression showed resistance to nucleic acid-target antibiotics, but sensitivity to cell wall-target drugs, yet long-term expression arrests growth. According to transcriptome investigation, the deletion of MSMEG_4236 upregulated all division and cell wall cluster operon genes, several mycolic acids and arabinogalactan biosynthesis genes and downregulated numerous promoters outside of the dcw cluster operon through binding at AAAGTG[G/T] sequence motifs. Conclusion: This study shows that the mycobacterial Rv2166c represses the division and cell wall cluster operon as a transcriptional regulator. Thus, modulating this gene's expression affects many mycobacteria phenotypes and environmental stress resistance. Several AAAGTG[G/T] motifs containing promoters have been found, demonstrating that Rv2166c regulates genes other than its operon. These data suggest that the Rv2166c can be targeted as future antituberculosis medicines.

Dataset Information

The Serine/Threonine kinase PrkC of Clostridioides difficile regulates export of CD1135, a septal peptidoglycan hydrolase

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