Engineering a super-secreting strain of Escherichia coli by directed co-evolution of the multiprotein Tat translocation machinery

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed co-evolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three super-secreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than that observed for wildtype TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of highly efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

Identification and in vitro Characterization of a Novel Phage Endolysin that Targets Gram-Negative Bacteria

Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis.

A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways

The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.

A unifying mechanism for the biogenesis of prokaryotic membrane proteins co-operatively integrated by the Sec and Tat pathways

The vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of the Streptomyces coelicolor Rieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.