title: The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity.
abstract: The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Frain
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Jones
authors.affiliation: Biopharmaceutical Development, MedImmune LLC, Gaithersburg, MD 20878, USA.
authors.forename: Ronald
authors.initials: R
authors.lastname: Schoner
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Walker
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom. Electronic address: c.robinson-504@kent.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; C000614170:Interferon-alpha2b; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies; C408585:twin-arginine translocase complex, E coli; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase
keywords: Bacillus subtilis; Signal peptide; Tat; TatAdCd; Twin-arginine