Role of the <i>Escherichia coli</i> Tat pathway in outer membrane integrity

Ize, Bérengère; Stanley, Nicola R.; Buchanan, Grant; Palmer, Tracy

doi:10.1046/j.1365-2958.2003.03504.x

Cited by 204 publications

(256 citation statements)

References 47 publications

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“…We suggest an additional benefit is to help coordinate constriction of the outer membrane with inward growth of the PG layer. Hydrolysis of septal PG is required for proper invagination of the outer membrane, which in turn is required for resistance to bile salts and other noxious compounds (9,43,44). Tethering FtsN to denuded glycans could help prevent septal PG synthesis from running too far ahead of the amidases that are needed for maintaining outer membrane integrity during cytokinesis.…”

Section: Discussionmentioning

confidence: 99%

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Yahashiri

Jorgenson

Weiss

2015

Proc. Natl. Acad. Sci. U.S.A.

142

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Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to "denuded" glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division.A defining feature of bacteria is the peptidoglycan (PG) cell wall or "sacculus" that confers cell shape, protects the cell against lysis caused by turgor pressure, and is the ultimate target of many clinically relevant antibiotics, including β-lactams and vancomycin (1-4). The structure of PG is characterized by a network of glycan strands connected at regular intervals by oligopeptide cross-links (Fig. 1). The glycans are built from a repeating disaccharide of β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), from which branch the oligopeptides that mediate cross-linking. During cell division, a new PG wall is assembled between the incipient daughter cells (5, 6), and subsequent cell separation depends on the activity of a collection of PG hydrolases that cleave various bonds in the PG mesh (7).In the model Gram-negative bacterium Escherichia coli, daughter-cell separation is driven primarily by three cell-wall amidases (8-10). These enzymes cleave the amide bond that joins the lactyl group of the MurNAc to the α-amino group of the first amino acid (L-Ala) of the stem peptide, releasing as products free oligopeptides and "denuded" glycan strands (Fig. 1). Lytic transglycosylases (LTs) that cleave the MurNAc-GlcNAc glycosidic bond and endopeptidases that cleave stem peptides make minor but still significant contributions to daughter-cell separation (9, 11). In Bacillus subtilis, a model Gram-positive species, the enzymology of daughter-cell separation is not as well characterized. The...

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Section: Discussionmentioning

confidence: 99%

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Yahashiri

Jorgenson

Weiss

2015

Proc. Natl. Acad. Sci. U.S.A.

142

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“…Wild-type E. coli strain MC4100 and isogenic ⌬tatC derivatives of MC4100, namely B1LK0 (53) or BMI1 (35), were used for all experiments except those involving E. coli PhoA, where strains DHB4, FÅ113, and FUDDY (30,38) were used instead. For all plasmids used in this study, see Table S2, and for details regarding their construction and the construction of the plasmid libraries, see SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Waraho

DeLisa

2009

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a reliable genetic selection strategy for isolating interacting proteins based on the ''hitchhiker'' mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an Nterminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 ␤-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer ␤-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.ligand binding proteins ͉ protein folding quality control ͉ signal peptide ͉ twin-arginine translocation ͉ 2-hybrid system P rotein-protein interactions are key molecular events that integrate multiple gene products into functional complexes in virtually every cellular process. Because such interactions mediate numerous disease states and biological mechanisms underlying the pathogenesis of bacterial and viral infections, identification of protein-protein interactions remains one of the most important challenges in the postgenomics era. The yeast 2-hybrid (Y2H) system (1) has been the tool of choice for revealing numerous protein-protein interactions, underlying diverse protein networks and complex protein machinery inside living cells. To date, Y2H has been used to generate protein interaction maps for humans (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), Saccharomyces cerivisiae (5, 6), vaccinia virus (7), and Escherichia coli bacteriophage T7 (8). Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (9, 10), and in certain instances ablate protein functi...

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“…5b). Morphological differences have been reported for tat mutants in other bacterial species (Ize et al, 2003;Kimura et al, 2006;Stanley et al, 2001).…”

Section: Prediction and Expression Of Potentialmentioning

confidence: 88%

“…Complementation analysis of B. bacteriovorus tat gene product function in E. coli E. coli tat mutants lose the ability to grow on LB agar plates containing 2 % SDS (Buchanan et al, 2002;Ize et al, 2003) due to the failure to transport Tat substrates, including AmiAC, which are required for maintenance of the cell envelope (Bernhardt & de Boer, 2003). E. coli tat mutants expressing similarly functional tat genes would show complementation by a restoration of growth on plates containing SDS.…”

Section: Rt-pcrmentioning

confidence: 99%

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

et al. 2011

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Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron–sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

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Role of the Escherichia coli Tat pathway in outer membrane integrity

Cited by 204 publications

References 47 publications

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

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