Tat Pathway Research Articles

Twin-Arginine Protein Translocation.

Twin-arginine protein translocation systems (Tat) translocate fully folded and co-factor-containing proteins across biological membranes. In this review, we focus on the Tat pathway of Gram-positive bacteria. The minimal Tat pathway is composed of two components, namely a TatA and TatC pair, which are often complemented with additional TatA-like proteins. We provide overviews of our current understanding of Tat pathway composition and mechanistic aspects related to Tat-dependent cargo protein translocation. This includes Tat pathway flexibility, requirements for the correct folding and incorporation of co-factors in cargo proteins and the functions of known cargo proteins. Tat pathways of several Gram-positive bacteria are discussed in detail, with emphasis on the Tat pathway of Bacillus subtilis. We discuss both shared and unique features of the different Gram-positive bacterial Tat pathways. Lastly, we highlight topics for future research on Tat, including the development of this protein transport pathway for the biotechnological secretion of high-value proteins and its potential applicability as an antimicrobial drug target in pathogens.

Plant mitochondria contain the protein translocase subunits TatB and TatC.

Twin-arginine translocation (Tat) pathways have been well-characterized in bacteria and chloroplasts. Genes encoding a TatC protein are found in almost all plant mitochondrial genomes but to date these have not been extensively investigated. For the first time it could be demonstrated that this mitochondrial-encoded TatC is a functional gene that is translated into a protein in the model plant Arabidopsis thaliana A TatB--like subunit localized to the inner membrane was also identified that is nuclear-encoded and is essential for plant growth and development, indicating that plants potentially require a Tat pathway for mitochondrial biogenesis.

Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Construction of a novel twin-arginine translocation (Tat)-dependent type expression vector for secretory production of heterologous proteins in Corynebacterium glutamicum

Corynebacterium glutamicum is recognized as a favorable host for the secretory production of heterologous proteins. However, there are few secretion-type expression vectors available for protein production in C. glutamicum. In this study, we constructed a shuttle expression vector pAU3, which harbors the strong promoter tac-M for constitutive gene transcription, the consensus RBS sequence for protein translation, and the strong cgR_0949 signal sequence for protein secretion via the Tat pathway in C. glutamicum. The applicability of pAU3 was confirmed by the highly efficient expression and secretion of the CAT protein in C. glutamicum. The vector pAU3 is highly useful for secretory production of heterologous proteins in C. glutamicum.

The Tat pathway as a biotechnological tool for the expression and export of heterologous proteins inEscherichia coli

Approximately 30% of all currently licensed, recombinantly expressed biotherapeutic products are produced in Escherichia coli, among which a significant proportion are exported to the periplasm by the general Secretory ‘Sec’ pathway. However, this pathway cannot handle many target proteins and the Tat pathway is emerging as a powerful alternative means of export. The Tat system exports fully folded proteins, moreover it preferentially exports correctly folded proteins and has been shown to export a range of biotherapeutics. This review will discuss our current understanding of the Tat pathway and its potential application for the industrial-scale production of biotherapeutics.

A Tat ménage à trois — The role of Bacillus subtilis TatAc in twin-arginine protein translocation

The twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy–TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization.

Exploring the Feasibility of the Sec Route to Secrete Proteins Using the Tat Route in Streptomyces lividans.

Streptomyces lividans uses mainly two pathways to target secretory proteins to the cytoplasmic membrane. The major pathway (Sec pathway) transports pre-proteins using the signal recognition particle, and the minor Tat pathway is responsible for the secretion using a folded conformation of a relatively low number of proteins. The signal peptides of the Sec-dependent alpha-amylase and the Tat-dependent agarase were interchanged and fused in-frame to the corresponding mature part of the other enzyme. Alpha-amylase was unable to use the Tat route when fused to the agarase signal peptide, while agarase used the Sec route when it was targeted by the alpha-amylase signal peptide. In addition to the signal peptide some yet unidentified parts of the secreted proteins may play a role in selecting the secretory route. Structure predictions for the Tat- and Sec-dependent proteins suggest that less structured proteins are more likely to be candidates for the Tat route.

Periplasmic Export of Bile Salt Hydrolase in Escherichia coli by the Twin-Arginine Signal Peptides

Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholesterolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained, E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21 ± 0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

SummaryThe Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in E scherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

Commentary: The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes.

Bacteria secrete macromolecules to manipulate their surrounding environment (abiotic or biotic one) for their own benefit. Among them, the diderms (Gram-negative), face the challenge of protein transport across an additional, important barrier: the outer membrane (OM). Specific pathways have evolved to transfer lipidated polypeptide chains to the OM, for example, the Lol system (Okuda and Tokuda, 2011). These polypeptides are then inserted to the inner OM leaflet via their N-terminal-attached lipid moiety. Some of them adopt a β-barrel fold or polymerize in a specific way to accomplish their complete insertion in the membrane. A large family of protein domains, named secretins (Figure ​(Figure1;1; Korotkov et al., 2011), that polymerize to form a channel in the OM, is present in many bacterial secretion systems, including the Type II Secretion System (T2SS), the Type IV pili (both T4aP and T4bP types) and the Type III Secretion System (T3SS). Figure 1 Protein domain organization of secretins and pilotins from Type II, Type III Secretion Systems, Type IV pili and the Tad system. Secretins of the Rhizobium (Rhc-) family of T3SSs, represented here from subgroup I Rhc-T3SS of Mesorhizobium loti and subgroup ... In a generalized scheme, the nascent polypeptide of a secretin carries an N-terminal signal that is recognized by the general secretion pathway (Sec). The Sec system transfers the polypeptide to the periplasmic space. Once there, most often an escorting lipoprotein, pilotin, or docking protein that is also targeted to the periplasmic space via the Sec or Tat pathways will assist with delivery, insertion and/or right polymerization of the polypeptide in the OM, most likely through enrollment of the Lol system (Collin et al., 2011; Okuda and Tokuda, 2011). Pilotins are extremely diverse in their folding and way of action, while they are usually specific to their cognate secretins (Figure ​(Figure1;1; Koo et al., 2012; Dunstan et al., 2013). In most cases, they are encoded within gene clusters that also encode their cognate secretins, as well as other subunits of the respective bacterial secretion system they serve. Type III secretion systems evolved for the direct translocation of bacterial proteins from the bacterial cytoplasm to the eukaryotic cell cytoplasm (Soto et al., 2006; Tampakaki et al., 2010; Marteyn et al., 2012; Tampakaki, 2014), a process involved in either pathogenesis or symbiosis with higher organisms. These systems appear to have evolved from the bacterial flagellum through gene loss and new gene acquisitions. Several modern T3SS families exist today that are specialized in the invasion of a broad range of hosts, animals or plants (Abby and Rocha, 2012). The bacterial flagellum does not possess a secretin while a T2SS/T4P originated secretin has been early acquired and evolved to a specialized T3SS secretin. However, in the case of the T3SS of Rhizobia (Rhc-T3SS), dedicated to symbiosis, this early acquired T2SS/T4P related secretin was lost and only a shorter aminoterminal remnant of this gene (named rhcC1), is present in the T3SS gene cluster (Figure ​(Figure1),1), while an acquisition of a Tad (Tight Adherence)—like secretin (named rhcC2) took place, possibly through DNA recombination events (Figure ​(Figure1;1; Abby and Rocha, 2012). The work of Mercante et al. (2015) nicely complements these findings by showing that a predicted lipoprotein (y4yS) of the Mesorhizobium loti Rhc-T3SS could act as a potential pilotin by influencing the level of RhcC2 protein present in bacterial membranes, while sharing common characteristics (tetatricopeptide repeats, TPRs) of the Tad system pilotins. Surprisingly, TPR domains have not been observed in other known pilotins within the remaining T3SS families, while they are present in Tad and T4aP pilotins (Figure ​(Figure1).1). This new finding, raised new questions. The mechanism by which Y4yS is piloting the Rhc-secretin to the OM still remains unknown. Furthermore, y4yS homologs are only present in a subset of Rhc-T3SSs (Gazi et al., 2012). Rhc-T3SS subgroup II clusters do not harbor a y4yS gene, while Rhc-T3SS subgroup III of Rhizobium etli although maintaining a rhcC1 gene, seems to have lost or never acquired a Tad like secretin (rhcC2), adding even more mystery to how this particular T3SS nanomachine transverses the OM. Why is a system-specific, non-conserved pilotin needed for the delivery and accommodation of a highly conserved domain in the outer bacterial membrane? This question cannot be answered as of this time, but it seems quite possible that more studies will further reveal this complexity before giving us some first clues.

Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis.

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.

Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc1 complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.

Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct Tat-Dependent Secretion of Methyl Parathion Hydrolase

The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

Proteomic Analysis of the Secretome of Haloarchaeon Natrinema sp. J7–2

Although in silico predictions have revealed that haloarchaea can be distinguished from other organisms in that the Tat pathway is used more extensively than the Sec pathway for haloarchaeal protein secretion, only a few haloarchaeal-secreted proteins have been experimentally confirmed. Here, the culture supernatant and membrane fraction of the haloarchaeon Natrinema sp. J7-2 grown at 23% salt concentration were subjected to RPLC-ESI-MS/MS analysis. In total, 46 predicted Tat substrates, 14 predicted Sec substrates, and 3 class III signal peptide-bearing proteins were detected. Approximately 65% of the detected Tat substrates contain lipoboxes, emphasizing the role of the Tat pathway in haloarchaeal lipoprotein secretion. Most of the detected Tat substrates are extracellular substrate (solute)-binding proteins and redox proteins. Despite the small number of Sec substrates, two of them, a cell surface glycoprotein and a putative lipoprotein carrier protein, were identified to be high-abundance secreted proteins. While limited proteins were detected in the culture supernatant, most of the secreted proteins were found in the membrane fraction. The anchoring of secreted proteins to the cell surface via a lipobox or a PGF-CTERM seems to be an adaptation strategy of haloarchaea to handle the harsh extracellular environment. Additionally, ∼15% of the integral membrane proteins (IMPs) detected in the membrane fraction possess putative Sec signal peptides or signal anchors, implying that the Sec pathway is important for membrane insertion of IMPs. This is the first report to describe the experimental secretome of haloarchaea and provide new information for better understanding of haloarchaeal protein secretion patterns.

Structure to function prediction of hypothetical protein KPN_00953 (Ycbk) from Klebsiella pneumoniae MGH 78578 highlights possible role in cell wall metabolism

BackgroundKlebsiella pneumoniae plays a major role in causing nosocomial infection in immunocompromised patients. Medical inflictions by the pathogen can range from respiratory and urinary tract infections, septicemia and primarily, pneumonia. As more K. pneumoniae strains are becoming highly resistant to various antibiotics, treatment of this bacterium has been rendered more difficult. This situation, as a consequence, poses a threat to public health. Hence, identification of possible novel drug targets against this opportunistic pathogen need to be undertaken. In the complete genome sequence of K. pneumoniae MGH 78578, approximately one-fourth of the genome encodes for hypothetical proteins (HPs). Due to their low homology and relatedness to other known proteins, HPs may serve as potential, new drug targets.ResultsSequence analysis on the HPs of K. pneumoniae MGH 78578 revealed that a particular HP termed KPN_00953 (YcbK) contains a M15_3 peptidases superfamily conserved domain. Some members of this superfamily are metalloproteases which are involved in cell wall metabolism. BLASTP similarity search on KPN_00953 (YcbK) revealed that majority of the hits were hypothetical proteins although two of the hits suggested that it may be a lipoprotein or related to twin-arginine translocation (Tat) pathway important for transport of proteins to the cell membrane and periplasmic space. As lipoproteins and other components of the cell wall are important pathogenic factors, homology modeling of KPN_00953 was attempted to predict the structure and function of this protein. Three-dimensional model of the protein showed that its secondary structure topology and active site are similar with those found among metalloproteases where two His residues, namely His169 and His209 and an Asp residue, Asp176 in KPN_00953 were found to be Zn-chelating residues. Interestingly, induced expression of the cloned KPN_00953 gene in lipoprotein-deficient E. coli JE5505 resulted in smoother cells with flattened edges. Some cells showed deposits of film-like material under scanning electron microscope.ConclusionsWe postulate that KPN_00953 is a Zn metalloprotease and may play a role in bacterial cell wall metabolism. Structural biology studies to understand its structure, function and mechanism of action pose the possibility of utilizing this protein as a new drug target against K. pneumoniae in the future.

Efficient export of prefolded, disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni.

This work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.