Dataset Information

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

ABSTRACT: Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal ? barrel domain ("? domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the ? domain is integrated into the outer membrane and the relationship between ? domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the ? barrel assembly machinery (Bam) complex interact with the ? domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire ? domain, BamA, BamB, and BamD subsequently interacted with discrete ? domain regions. BamB and BamD remained bound to the ? domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of ? domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of ? barrel proteins.

SUBMITTER: Ieva R

PROVIDER: S-EPMC3150907 | biostudies-other | 2011 Aug

REPOSITORIES: biostudies-other

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Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

Ieva Raffaele R Tian Pu P Peterson Janine H JH Bernstein Harris D HD

Proceedings of the National Academy of Sciences of the United States of America 20110606 31

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the β domain is integrated into the outer membrane and the relationship between β domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of t ...[more]

PMID: 21646511

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Project description:At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCE Bacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

Dataset Information

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

Publications

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

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