The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function

Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate

A three-way comparative genomic analysis of Mannheimia haemolytica isolates

<p>Abstract</p> <p>Background</p> <p><it>Mannhemia haemolytica </it>is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of <it>M. haemolytica </it>at molecular level and assist in devising an effective control strategy.</p> <p>Description</p> <p>During our comparative genomic sequence analysis of three <it>Mannheimia haemolytica </it>isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors.</p> <p>Conclusions</p> <p>During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in <it>M. haemolytica </it>will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.</p

Genes Required for Growth at High Hydrostatic Pressure in Escherichia coli K-12 Identified by Genome-Wide Screening

Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo. The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure

Transport of Folded Proteins by the Tat System

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations

Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein.

Cleavable N-terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec-type or delta pH-driven protein translocase. The targeting signals specify choice of translocation pathway, yet all resemble typical bacterial 'signal' peptides in possessing a charged N-terminus (N-domain), hydrophobic core region (H-domain) and more polar C-terminal region (C-domain). We have previously shown that a twin-arginine motif in the N-domain is essential for targeting by the delta pH-dependent pathway, but it has remained unclear why targeting signals for this system (transfer peptides) are not recognized by the Sec apparatus. We show here that the conserved charge distribution around the H-domain in the 23K transfer peptide (twin-Arg in the N-domain, Lys in the C-domain) constitutes a 'Sec-avoidance' signal. The C-domain Lys, while not important for delta pH-dependent targeting, is the only barrier to Sec-dependent translocation; its removal generates an apparently perfect signal peptide. Conversely, insertion of twin-Arg into the N-domain of a Sec substrate has little effect, as has insertion of a C-domain Lys, but the combined substitutions almost totally block transport. We also show that the 23K mature protein is incapable of being targeted by the Sec pathway, and it is proposed that the role of the Sec-avoidance motif in the transfer peptide is to prevent futile interactions with the Sec apparatus

Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein