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In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions.

ABSTRACT: The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.

SUBMITTER: Bouveret E

PROVIDER: S-EPMC103764 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

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In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions.

Bouveret E E Bénédetti H H Rigal A A Loret E E Lazdunski C C

Journal of bacteriology 19991001 20

The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to t ...[more]

PMID: 10515919

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Project description:Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun's lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun's lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

Project description:Grafting is an ancient plant propagation technique widely used in horticultural crops, particularly in fruit trees. However, the involvement of two different species in grafting may lead to lack of affinity and severe disorders between the graft components, known as graft-incompatibility. This complex agronomic trait is traditionally classified into two categories: "localized" (weak graft unions with breaks in cambial and vascular continuity at the graft interface and absence of visual symptoms in scion leaves and shoots) and "translocated" (degeneration of the sieve tubes and phloem companion cells at the graft interface causing translocation problems in neighboring tissues, and reddening/yellowing of scion leaves). Over the decades, more attention has been given to the different mechanisms underlying the "localized" type of graft-incompatibility; whereas the phenylpropanoid-derived compounds and the differential gene expression associated with the "translocated" graft-incompatibility remain unstudied. Therefore, the aim of this study was to shed light on the biochemical and molecular mechanisms involved in the typical "translocated" graft-incompatibility of peach/plum graft-combinations. In this study, the "Summergrand" (SG) nectarine cultivar was budded on two plum rootstocks: "Adara" and "Damas GF 1869". "Translocated" symptoms of incompatibility were shown and biochemically characterized in the case of "SG/Damas GF 1869" graft-combination, 3 years after grafting. Non-structural carbohydrates (soluble sugars and starch), phenolic compounds and antioxidant activity, were significantly enhanced in the incompatible graft-combination scion. Similarly, the enzymatic activities of the antioxidant enzyme peroxidase, the phenylalanine ammonia-lyase (PAL) and polyphenol oxidase involved in the phenylpropanoid pathway were significantly affected by the incompatible rootstock "Damas GF 1869", inducing higher activities in the scion than those induced by the compatible rootstock "Adara". In addition, a positive and strong correlation was obtained between total phenol content, antioxidant capacity and the expression of the key genes involved in the phenylpropanoid pathway, PAL1 and PAL2. Regarding the "SG/Adara" graft-combination, there were neither external symptoms of "translocated" incompatibility nor significant differences in the biochemical and molecular parameters between scion and rootstock, proving it to be a compatible combination. The differential expression of PAL genes together with the biochemical factors cited above could be good markers for the "translocated" peach/plum graft-incompatibility.

Dataset Information

In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions.

Publications

In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions.

Similar Datasets

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