Tracy Palmer | University of Dundee (original) (raw)

Papers by Tracy Palmer

Research paper thumbnail of Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure

European Journal of Biochemistry, 2001

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of ... more The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

Research paper thumbnail of Oligomeric Properties and Signal Peptide Binding by Escherichia coli Tat Protein Transport Complexes

Journal of Molecular Biology, 2002

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded... more The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

Research paper thumbnail of Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels: Native Tat complexes

Research paper thumbnail of Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

Research paper thumbnail of Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

Molecular Microbiology, 1996

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopt... more The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCI2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but overexpression of MobB increases the level of factor X.

Research paper thumbnail of Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit . Identification of a soluble precursor of the small subunit in a hypB mutant

European Journal of Biochemistry, 1998

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the peri... more An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal trypticfragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416Ϫ4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Research paper thumbnail of Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion

Molecular Microbiology, 2015

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA... more BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Research paper thumbnail of Formation of functional Tat translocases from heterologous components

BMC microbiology, 2006

The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the th... more The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components. All three subunits of the Tat system from the Gram positive organism Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed ...

Research paper thumbnail of Bacterial formate hydrogenlyase complex

Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2014

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimat... more Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor t...

Research paper thumbnail of 1 Bacterial flagellar diversity and evolution: seek simplicity and distrust it? 6 Interchangeable modules in bacterial thiol-disulfide exchange pathways

Research paper thumbnail of PROTON-TRANSLOCATING TRANSHYDROGENASE IN MEMBRANES OF PHOTOSYNTHETIC BACTERIA

Research paper thumbnail of Single-molecule fluorescence microscopy of the twin-arginine translocation (Tat) system

Research paper thumbnail of Reversible assembly of twin-arginine protein translocation sites

Research paper thumbnail of Protein targeting by the bacterial twin-arginine translocation (Tat) pathway

Current Opinion in Microbiology, 2005

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane ... more The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.

Research paper thumbnail of Escherichia coli Strains Blocked in Tat-Dependent Protein Export Exhibit Pleiotropic Defects in the Cell Envelope

Journal of Bacteriology, 2001

The Tat system is a recently discovered protein export pathway that serves to translocate folded ... more The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by

Research paper thumbnail of The Tat protein translocation pathway and its role in microbial physiology

Advances in Microbial Physiology, 2003

The Tat (twin arginine translocation) protein transport system functions to export folded protein... more The Tat (twin arginine translocation) protein transport system functions to export folded protein substrates across the bacterial cytoplasmic membrane and to insert certain integral membrane proteins into that membrane. It is entirely distinct from the Sec pathway. Here, we describe our current knowledge of the molecular features of the Tat transport system. In addition, we discuss the roles that the

Research paper thumbnail of The Tat protein export pathway

Molecular Microbiology, 2000

Summary The Tat ( twin-arginine translocation) system is a bac- terial protein export pathway wit... more Summary The Tat ( twin-arginine translocation) system is a bac- terial protein export pathway with the remarkable ability to transport folded proteins across the cyto- plasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a character- istic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat

Research paper thumbnail of A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens

The Journal of Cell Biology, 2014

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secret... more Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

Research paper thumbnail of Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit . Identification of a soluble precursor of the small subunit in a hypB mutant

European Journal of Biochemistry, 1998

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the peri... more An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal trypticfragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416Ϫ4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Research paper thumbnail of TatD Is a Cytoplasmic Protein with DNase Activity

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twi... more The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni 2؉ or Zn 2؉ affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.

Research paper thumbnail of Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure

European Journal of Biochemistry, 2001

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of ... more The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

Research paper thumbnail of Oligomeric Properties and Signal Peptide Binding by Escherichia coli Tat Protein Transport Complexes

Journal of Molecular Biology, 2002

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded... more The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

Research paper thumbnail of Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels: Native Tat complexes

Research paper thumbnail of Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

Research paper thumbnail of Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

Molecular Microbiology, 1996

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopt... more The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCI2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but overexpression of MobB increases the level of factor X.

Research paper thumbnail of Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit . Identification of a soluble precursor of the small subunit in a hypB mutant

European Journal of Biochemistry, 1998

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the peri... more An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal trypticfragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416Ϫ4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Research paper thumbnail of Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion

Molecular Microbiology, 2015

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA... more BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Research paper thumbnail of Formation of functional Tat translocases from heterologous components

BMC microbiology, 2006

The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the th... more The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components. All three subunits of the Tat system from the Gram positive organism Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed ...

Research paper thumbnail of Bacterial formate hydrogenlyase complex

Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2014

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimat... more Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor t...

Research paper thumbnail of 1 Bacterial flagellar diversity and evolution: seek simplicity and distrust it? 6 Interchangeable modules in bacterial thiol-disulfide exchange pathways

Research paper thumbnail of PROTON-TRANSLOCATING TRANSHYDROGENASE IN MEMBRANES OF PHOTOSYNTHETIC BACTERIA

Research paper thumbnail of Single-molecule fluorescence microscopy of the twin-arginine translocation (Tat) system

Research paper thumbnail of Reversible assembly of twin-arginine protein translocation sites

Research paper thumbnail of Protein targeting by the bacterial twin-arginine translocation (Tat) pathway

Current Opinion in Microbiology, 2005

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane ... more The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.

Research paper thumbnail of Escherichia coli Strains Blocked in Tat-Dependent Protein Export Exhibit Pleiotropic Defects in the Cell Envelope

Journal of Bacteriology, 2001

The Tat system is a recently discovered protein export pathway that serves to translocate folded ... more The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by

Research paper thumbnail of The Tat protein translocation pathway and its role in microbial physiology

Advances in Microbial Physiology, 2003

The Tat (twin arginine translocation) protein transport system functions to export folded protein... more The Tat (twin arginine translocation) protein transport system functions to export folded protein substrates across the bacterial cytoplasmic membrane and to insert certain integral membrane proteins into that membrane. It is entirely distinct from the Sec pathway. Here, we describe our current knowledge of the molecular features of the Tat transport system. In addition, we discuss the roles that the

Research paper thumbnail of The Tat protein export pathway

Molecular Microbiology, 2000

Summary The Tat ( twin-arginine translocation) system is a bac- terial protein export pathway wit... more Summary The Tat ( twin-arginine translocation) system is a bac- terial protein export pathway with the remarkable ability to transport folded proteins across the cyto- plasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a character- istic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat

Research paper thumbnail of A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens

The Journal of Cell Biology, 2014

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secret... more Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

Research paper thumbnail of Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit . Identification of a soluble precursor of the small subunit in a hypB mutant

European Journal of Biochemistry, 1998

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the peri... more An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal trypticfragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416Ϫ4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Research paper thumbnail of TatD Is a Cytoplasmic Protein with DNase Activity

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twi... more The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni 2؉ or Zn 2؉ affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.