Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell.

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Identity Determinants of the Translocation Signal for a Type 1 Secretion System.

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process.

Type I Secretion Systems-One Mechanism for All?

Type I secretion systems (T1SS) are widespread in Gram-negative bacteria, especially in pathogenic bacteria, and they secrete adhesins, iron-scavenger proteins, lipases, proteases, or pore-forming toxins in the unfolded state in one step across two membranes without any periplasmic intermediate into the extracellular space. The substrates of T1SS are in general characterized by a C-terminal secretion sequence and nonapeptide repeats, so-called GG repeats, located N terminal to the secretion sequence. These GG repeats bind Ca2+ ions in the extracellular space, which triggers folding of the entire protein. Here we summarize our current knowledge of how Gram-negative bacteria secrete these substrates, which can possess a molecular mass of up to 1,500 kDa. We also describe recent findings that demonstrate that the absence of periplasmic intermediates, the "classic" mode of action, does not hold true for all T1SS and that we are beginning to realize modifications of a common theme.

Type I secretion system-it takes three and a substrate.

Type I secretion systems are widespread in Gram-negative bacteria and mediate the one-step translocation of a large variety of proteins serving for diverse purposes, including nutrient acquisition or bacterial virulence. Common to most substrates of type I secretion systems is the presence of a C-terminal secretion sequence that is not cleaved during or after translocation. Furthermore, these protein secretion nanomachineries are always composed of an ABC transporter, a membrane fusion protein, both located in the inner bacterial membrane, and a protein of the outer membrane. These three membrane proteins transiently form a 'tunnel channel' across the periplasmic space in the presence of the substrate. Here we summarize the recent findings with respect to structure, function and application of type I secretion systems.

Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc.