Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of
            <i>Aeromonas hydrophila</i>

Schoenhofen, Ian C.; Li, Gang; Strozen, Timothy G.; Howard, S. Peter

doi:10.1128/jb.187.18.6370-6378.2005

Cited by 19 publications

(21 citation statements)

References 54 publications

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“…novicidia uses a T2SS that even lacks the GspLMC components to export chitinases, proteinases, and ␤-glucosidases (17). The presence of two ATPases in E. minutum, which are typical for T4P, does not necessarily argue against a T2SS; the T2SS of Aeromonas hydrophila also has two ATPases, and they are thought to increase the efficiency of the secretory process (47).…”

Section: Vol 75 2009 Genomic Analysis Of Elusimicrobium Minutum 2845mentioning

confidence: 99%

Genomic Analysis of “ Elusimicrobium minutum ,” the First Cultivated Representative of the Phylum “ Elusimicrobia ” (Formerly Termite Group 1)

Herlemann

Geissinger

Ikeda-Ohtsubo

et al. 2009

Appl Environ Microbiol

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Organisms of the candidate phylum termite group 1 (TG1) are regularly encountered in termite hindguts but are present also in many other habitats. Here, we report the complete genome sequence (1.64 Mbp) of "Elusimicrobium minutum" strain Pei191 T , the first cultured representative of the TG1 phylum. We reconstructed the metabolism of this strictly anaerobic bacterium isolated from a beetle larva gut, and we discuss the findings in light of physiological data. E. minutum has all genes required for uptake and fermentation of sugars via the Embden-Meyerhof pathway, including several hydrogenases, and an unusual peptide degradation pathway comprising transamination reactions and leading to the formation of alanine, which is excreted in substantial amounts. The presence of genes encoding lipopolysaccharide biosynthesis and the presence of a pathway for peptidoglycan formation are consistent with ultrastructural evidence of a gram-negative cell envelope. Even though electron micrographs showed no cell appendages, the genome encodes many genes putatively involved in pilus assembly. We assigned some to a type II secretion system, but the function of 60 pilE-like genes remains unknown. Numerous genes with hypothetical functions, e.g., polyketide synthesis, nonribosomal peptide synthesis, antibiotic transport, and oxygen stress protection, indicate the presence of hitherto undiscovered physiological traits. Comparative analysis of 22 concatenated single-copy marker genes corroborated the status of "Elusimicrobia" (formerly TG1) as a separate phylum in the bacterial domain, which was so far based only on 16S rRNA sequence analysis.At least half of the phylum-level lineages within the domain Bacteria do not comprise pure cultures but are, rather, represented only by 16S rRNA gene sequences of environmental origin (43). The number of such candidate phyla is still growing, and the biology of the members of these phyla is usually completely obscure. The first sequences of the candidate phylum termite group 1 (TG1) (23) were obtained from the hindgut of the termite Reticulitermes speratus, where they represent a substantial portion of the gut microbiota (21, 41). Meanwhile, numerous sequences affiliated with this phylum have also been retrieved from habitats other than termite guts. They form several deep-branching lineages comprising sequences derived not only from intestinal tracts but also from soils, sediments, and contaminated aquifers (14,20).Recently, we were able to isolate strain Pei191 T , the first pure-culture representative of the TG1 phylum, from the gut of a humivorous scarab beetle larva, Pachnoda ephippiata (14). Based on the 16S rRNA gene sequence, strain Pei191T is a member of the "intestinal cluster," which consists of sequences derived from invertebrate guts and cow rumen (20) and is only distantly related to the so-called "endomicrobia," a lineage of TG1 bacteria comprising endosymbionts of termite gut protozoa (24,42,54). It is an obligately anaerobic ultramicrobacterium that grows heterotrophically on g...

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Section: Vol 75 2009 Genomic Analysis Of Elusimicrobium Minutum 2845mentioning

confidence: 99%

Genomic Analysis of “ Elusimicrobium minutum ,” the First Cultivated Representative of the Phylum “ Elusimicrobia ” (Formerly Termite Group 1)

Herlemann

Geissinger

Ikeda-Ohtsubo

et al. 2009

Appl Environ Microbiol

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“…The GspAB Ah complex has been demonstrated to be required for the localization and multimerization of the GspD secretin multimer in the outer membrane of A. hydrophila (3). We have also shown that the GspA Ah cytoplasmic domain is a novel ATPase (46) and have confirmed the function of a peptidoglycan-binding site in the GspA Ah periplasmic domain as being required for the assembly of the secretin multimer (17,25). Thus, our working hypothesis is that the GspAB complex in some way modifies or organizes the peptidoglycan to allow assembly of the GspD secretin, a function presumably necessitated by the 50-kDa size constraint imposed by the peptidoglycan mesh (9).…”

mentioning

confidence: 67%

“…6) in a dose-dependent manner. Since GspAB functions as a heteromultimeric complex (46), this phenotype was likely caused by a dominant negative effect of GspAB Vc whereby the assembly of an inactive heteromultimeric complex composed of GspAB Ah and GspAB Vc reduces the ability of the strain to assemble a functional secretin multimer and, thus, secrete T2SS substrates into the growth medium. The dominant negative effect of GspAB Vc expression is consistent with the ability to inactivate the function of a multimeric protein by the expression of a mutant subunit in trans, a hallmark of multisubunit proteins.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the GspAB Complex in Assembly of the Type II Secretion System Secretin of Aeromonas and Vibrio Species

Strozen

Stanley

et al. 2011

J Bacteriol

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The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA Ah , is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB Ah ). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD Ah ) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida, similar to that previously observed in A. hydrophila. In the Vibrionaceae species Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae (gspA Vc ) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae. In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans, further suggesting that GspAB Vc performs the same role in Vibrio species as GspAB Ah does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.

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“…The periplasmic domain of ExeA has a peptidoglycan (PG)-binding domain that has been shown to bind PG from A. hydrophila, Escherichia coli, and Bacillus subtilis (20). Interaction with PG results in multimerization of ExeA in vitro, and in vivo ExeAB assembles into large complexes of up to 12 monomers of each protein (20,21). Point mutations in conserved residues within the PG-binding domain of ExeA demonstrated that PG binding is required for ExeD assembly (20).…”

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Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System

et al. 2017

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In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the D,D-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCEThe PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG crosslinking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila. In a heterologous assembly model in E. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.KEYWORDS type II secretion system, peptidoglycan, secretin assembly T he secretion of protein toxins and enzymes via the type II secretion system (T2SS) is an important virulence mechanism as well as a major route of scavenging enzyme secretion and surface protein assembly for many Gram-negative bacteria (1-4). The T2SS is a large complex composed of 12 to 15 highly conserved proteins designated

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Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila

Cited by 19 publications

References 54 publications

Genomic Analysis of “ Elusimicrobium minutum ,” the First Cultivated Representative of the Phylum “ Elusimicrobia ” (Formerly Termite Group 1)

Genomic Analysis of “ Elusimicrobium minutum ,” the First Cultivated Representative of the Phylum “ Elusimicrobia ” (Formerly Termite Group 1)

Involvement of the GspAB Complex in Assembly of the Type II Secretion System Secretin of Aeromonas and Vibrio Species

Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System

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