Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator in
            <i>Pseudomonas aeruginosa</i>

Greene, Neil G.; Fumeaux, Coralie; Bernhardt, Thomas G.

doi:10.1073/pnas.1717925115

Cited by 41 publications

(58 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Without LpoB to induce and/or stabilise this state the frequency with which this sporadic activation occurs and/or the length of time PBP1B is active are not sufficient for the minimal PG synthesis threshold in the cell. This model is consistent with a previous report showing that limited PBP1B activity in the cell in the absence of LpoB is responsible for cell death (Markovski et al, ; Greene et al, ). R157A and E341A appear to be exceptions to this hypothesis as they have lower basal activities similar to R109G but retain similar affinity to LpoB as WT.…”

Section: Discussionsupporting

confidence: 93%

“…This supports a model in which activation of the synthase proceeds via a conformational change initiated by LpoB binding to its UB2H domain (Egan et al, ; Markovski et al, ). This hypothesis was further supported by the isolation of P. aeruginosa PBP1B versions with similar Lpo bypass mutations (Greene et al, ). However, the mechanism by which LpoB binding activates both PG synthesis activities simultaneously remained elusive.…”

Section: Introductionmentioning

confidence: 85%

See 1 more Smart Citation

Induced conformational changes activate the peptidoglycan synthase PBP1B

Egan

Maya-Martinez

Ayala

et al. 2018

Molecular Microbiology

View full text Add to dashboard Cite

SummaryBacteria surround their cytoplasmic membrane with an essential, stress‐bearing peptidoglycan (PG) layer consisting of glycan chains linked by short peptides into a mesh‐like structure. Growing and dividing cells expand their PG layer using inner‐membrane anchored PG synthases, including Penicillin‐binding proteins (PBPs), which participate in dynamic protein complexes to facilitate cell wall growth. In Escherichia coli, and presumably other Gram‐negative bacteria, growth of the mainly single layered PG is regulated by outer membrane‐anchored lipoproteins. The lipoprotein LpoB is required to activate PBP1B, which is a major, bi‐functional PG synthase with glycan chain polymerising (glycosyltransferase) and peptide cross‐linking (transpeptidase) activities. In this work we show how the binding of LpoB to the regulatory UB2H domain of PBP1B activates both activities. Binding induces structural changes in the UB2H domain, which transduce to the two catalytic domains by distinct allosteric pathways. We also show how an additional regulator protein, CpoB, is able to selectively modulate the TPase activation by LpoB without interfering with GTase activation.

show abstract

Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 85%

Induced conformational changes activate the peptidoglycan synthase PBP1B

Egan

Maya-Martinez

Ayala

et al. 2018

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…Transposon mutagenesis. P. aeruginosa PAO1 Δhfq cells were mutagenized with the minitransposon pBTK30 (59) using a mating protocol adapted from a previously described procedure (60). The donor strain SM10(pir) carrying pBTK30 was grown in LB supplemented with 50 g ml Ϫ1 ampicillin at 37°C, and PAO1 Δhfq was grown in NCE-succinate at 42°C overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Genomic DNA from thawed cell pellets of each mutant library was extracted, fragmented, poly(C) tailed, and purified as previously described (61). gDNA libraries were constructed and sequenced as previously described (60). Trimmed reads were mapped to TA insertion sites, normalized to total reads, and visualized using the Sanger Artemis genome browser and annotation tool as previously described (60).…”

Section: Methodsmentioning

confidence: 99%

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

et al. 2019

View full text Add to dashboard Cite

Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

show abstract

“…The phospholipid (PL) and LPS membrane components are synthesized in the cytosol and 67 within the inner leaflet of the IM. They must be transported across the IM and through the 68 periplasm to build the OM (for reviews, see (1,(4)(5)(6)). LPS consists of three covalently attached 69 groups: a glycolipid called Lipid A, a core oligosaccharide, and a longer, variable O-antigen 70 polysaccharide chain.…”

Section: Introduction 56mentioning

confidence: 99%

An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis inEscherichia coli

Fivenson

Bernhardt

2020

Preprint

Self Cite

View full text Add to dashboard Cite

22Gram-negative bacteria are surrounded by a complex cell envelope that includes two 24 membranes. The outer membrane prevents many drugs from entering these cells and is thus a 25 major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the 26 asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet 27 and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share a 28 common intermediate. Proper membrane biogenesis therefore requires that the flux through 29 each pathway be balanced. In Escherichia coli, a major control point in establishing this 30 balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are 31 controlled through its degradation by the inner membrane protease FtsH and its presumed 32 adaptor protein LapB(YciM). How turnover of LpxC is controlled has remained unclear for 33 many years. Here, we demonstrate that the essential protein of unknown function YejM(PbgA), 34 which we have renamed ClxD (control of LpxC degradation), participates in this regulatory 35 pathway. Suppressors of ClxD essentiality were identified in lpxC and lapB, and LpxC 36 overproduction was shown to be sufficient to allow survival of ΔclxD mutants. Furthermore, the 37 half-life of LpxC was shown to be reduced in cells lacking ClxD, and genetic and physical 38 interactions between LapB and ClxD were detected. Taken together, our results are consistent 39 with a model in which ClxD directly modulates LpxC turnover by FtsH-LapB to regulate LPS 40 synthesis and maintain membrane homeostasis. 41 3 SIGNIFICANCE 43The outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-44 negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the 45 synthesis of these lipid species must be balanced for the membrane to maintain its barrier 46 function in blocking drug entry. In this report, we identify an essential protein of unknown 47 function as a key new factor in maintaining LPS/phospholipid balance in the model bacterium 48Escherichia coli. Our results provide novel insight into how this organism and most likely other 49

show abstract

Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator in Pseudomonas aeruginosa

Cited by 41 publications

References 32 publications

Induced conformational changes activate the peptidoglycan synthase PBP1B

Induced conformational changes activate the peptidoglycan synthase PBP1B

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis inEscherichia coli

Contact Info

Product

Resources

About