SecA – a multidomain and multitask bacterial export protein

Most bacterial secretory proteins destined to the extracytoplasmic space are secreted posttranslationally by the Sec translocase. SecA, a key component of the Sec system, is the ATPase motor protein, directly responsible for transferring the preprotein across the cytoplasmic membrane. SecA is a large protein, composed of several domains, capable of binding client preproteins and a variety of partners, including the SecYEG inner membrane channel complex, membrane phospholipids and ribosomes. SecA-mediated translocation can be divided into two major steps: (1) targeting of the preproteins to the membrane translocation apparatus and (2) transport across the membrane through the SecYEG channel. In this review we present current knowledge regarding SecA structure and function of this protein in both translocation steps. The most recent model of the SecA-dependent preprotein mechanical translocation across the bacterial cytoplasmic membrane is described. A possibility of targeting SecA with inhibitory compounds as a strategy to combat pathogenic bacteria will be discussed as well.

Genome characterization of 'Candidatus Phytoplasma meliae' (isolate ChTYXIII)

Candidatus Phytoplasma meliae (subgroups 16SrXIII-G and XIII-C) has been reported in association to chinaberry yellowing disease in Argentina, Bolivia and Paraguay. In Argentina, this disease constitutes a major phytosanitary problem for chinaberry forestry production. To date, no genome information of this phytoplasma and others from 16SrXIII-group has been published, hindered its characterization at genomic level. Here we analyze the draft genome of Candidatus Phytoplasma meliae strain ChTYXIII obtained from a chinaberry-infected plant using a metagenomics approach. The draft assembly consists of twenty-one contigs with a total length of 751.949 bp. The annotation contains 669 CDSs, 34tRNA and one set of rRNA operons. Metabolic pathways analysis indicated that the ChTYXIII contains the complete core genes for glycolysis and functional sec system for translocation of proteins. The phylogenetic relationships inferred 132 single copy genes (orthologues core) analysis revealed that Ca. P. meliae constitutes a clade closely related to the Ca. australiense and Ca. P. solani. Thirty-one putative effectors were identified, among which a homologue to SAP11 was found and others that have only been described in this pathogen. This work provides relevant genomic information for Ca. P. meliae and constitutes the first genome described for the group 16SrXIII (MPV).

First Succinylome Profiling of Vibrio alginolyticus Reveals Key Role of Lysine Succinylation in Cellular Metabolism and Virulence

Recent studies have shown that a key strategy of many pathogens is to use post-translational modification (PTMs) to modulate host factors critical for infection. Lysine succinylation (Ksuc) is a major PTM widespread in prokaryotic and eukaryotic cells, and is associated with the regulation of numerous important cellular processes. Vibrio alginolyticus is a common pathogen that causes serious disease problems in aquaculture. Here we used the affinity enrichment method with LC-MS/MS to report the first identification of 2082 lysine succinylation sites on 671 proteins in V. alginolyticus, and compared this with the lysine acetylation of V. alginolyticus in our previous work. The Ksuc modification of SodB and PEPCK proteins were further validated by Co-immunoprecipitation combined with Western blotting. Bioinformatics analysis showed that the identified lysine succinylated proteins are involved in various biological processes and central metabolism pathways. Moreover, a total of 1,005 (25.4%) succinyl sites on 502 (37.3%) proteins were also found to be acetylated, which indicated that an extensive crosstalk between acetylation and succinylation in V. alginolyticus occurs, especially in three central metabolic pathways: glycolysis/gluconeogenesis, TCA cycle, and pyruvate metabolism. Furthermore, we found at least 50 (7.45%) succinylated virulence factors, including LuxS, Tdh, SodB, PEPCK, ClpP, and the Sec system to play an important role in bacterial virulence. Taken together, this systematic analysis provides a basis for further study on the pathophysiological role of lysine succinylation in V. alginolyticus and provides targets for the development of attenuated vaccines.

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity – a prerequisite for understanding how biological systems works – has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, ∼30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient – in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

A ‘proton ratchet’ for coupling the membrane potential to protein transport

The proton-motive force (PMF) – the electrochemical gradient of protons across energy-conserving membranes – powers protein transport in bacteria, mitochondria and chloroplasts. Here, we propose a ‘proton ratchet’ mechanism for this process. In the Sec system of bacteria, protons are stripped from lysine side chains of the pre-protein at the cytosolic face of the plasma membrane, then replaced on the exterior, aided by the pH component of the PMF (ΔpH; acidic outside). This gives the translocating region of pre-protein a net negative charge, promoting electrophoretic diffusion across the membrane driven by membrane-potential (ΔΨ; positive outside). For mitochondrial import (through the TIM23 complex) the proton ratchet acts in the opposite direction, with negatively charged residues protonated for passage across the inner membrane into the negative matrix. The proton ratchet is an elegant solution for coupling the PMF to transport, likely to be used by a range of other transporters of charged molecules.

SEC-SANS: size exclusion chromatography combinedin situwith small-angle neutron scattering

The first implementation and use of anin situsize exclusion chromatography (SEC) system on a small-angle neutron scattering instrument (SANS) is described. The possibility of deploying such a system for biological solution scattering at the Institut Laue–Langevin (ILL) has arisen from the fact that current day SANS instruments at ILL now allow datasets to be acquired using small sample volumes with exposure times that are often shorter than a minute. This capability is of particular importance for the study of unstable biological macromolecules where aggregation or denaturation issues are a major problem. The first use of SEC-SANS on ILL's instrument D22 is described for a variety of proteins including one particularly aggregation-prone system.