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Matthew P DeLisa

Publications and source records attributed to Matthew P DeLisa.

14 recordsLinked to original sources
PubMed2007

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides.

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Amino Acid Sequence↗
PubMed2006

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study.

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.

Algorithms↗
PubMed2006

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway.

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.

Amino Acid Sequence↗
PubMed2006

Collective bacterial dynamics revealed using a three-dimensional population-scale defocused particle tracking technique.

An ability to monitor bacterial locomotion and collective dynamics is crucial to our understanding of a number of well-characterized phenotypes including biofilm formation, chemotaxis, and virulence. Here, we report the tracking of multiple swimming Escherichia coli cells in three spatial dimensions and at single-cell resolution using a novel three-dimensional (3D) defocused particle tracking (DPT) method. The 3D trajectories were generated for wild-type Escherichia coli strain RP437 as well as for isogenic derivatives that display smooth swimming due to a cheA deletion (strain RP9535) or incessant tumbling behavior due to a cheZ deletion (strain RP1616). The 3D DPT method successfully differentiated these three modes of locomotion and allowed direct calculation of the diffusion coefficient for each strain. As expected, we found that the smooth swimmer diffused more readily than the wild type, and both the smooth swimmer and the wild-type cells exhibited diffusion coefficients that were at least two orders of magnitude larger than that of the tumbler. Finally, we found that the diffusion coefficient increased with increasing cell density, a phenomenon that can be attributed to the hydrodynamic disturbances caused by neighboring bacteria.

Bacterial Proteins↗
PubMed2005

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis.

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Cell Communication↗
PubMed2005

Twin-arginine translocation of active human tissue plasminogen activator in Escherichia coli.

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

Arginine↗
PubMed2005

Identification of a twin-arginine translocation system in Pseudomonas syringae pv. tomato DC3000 and its contribution to pathogenicity and fitness.

The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.

Anti-Bacterial Agents↗
PubMed2004

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

Amino Acid Motifs↗
PubMed2004

Dissecting the twin-arginine translocation pathway using genome-wide analysis.

A recently discovered route for protein export, known as the twin-arginine translocation (Tat) pathway, has received much attention owing to several atypical characteristics that distinguish it from other transport mechanisms. For instance, recent evidence has clearly established that this pathway only transports folded polypeptides. Moreover, several studies have demonstrated a vital role for the Tat pathway in bacterial pathogenesis. In this review, we discuss genomic approaches that have been employed to determine the prevalence and capacity of the Tat system and how the information generated from these approaches is helping to connect Tat transport to bacterial physiology and virulence.

Bacteria↗
PubMed2004

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway.

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.

Arabidopsis Proteins↗
PubMed2003

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway.

To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F(AB) antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F(AB) protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.

Alkaline Phosphatase↗
PubMed2002

Bacterial autoinduction: looking outside the cell for new metabolic engineering targets.

Recent evidence has demonstrated that cell-to-cell signaling is a fundamental activity carried out by numerous microorganisms. A number of specialized processes are reported to be regulated by density-dependent signaling molecules including antibiotic production, bioluminescence, biofilm formation, genetic competence, sporulation, swarming motility and virulence. However, a more centralized role for quorum sensing is emerging where quorum signaling pathways overlap with stress and starvation circuits to regulate cellular adaptation to changing environmental conditions. The interplay of these phenomena is especially critical in the expression of recombinant proteins where elicitation of stress responses can dramatically impact cellular productivity.

Journal Article↗
PubMed2002

Genetic analysis of the twin arginine translocator secretion pathway in bacteria.

The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.

Amino Acid Sequence↗