Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross‐linking enzyme microbial transglutaminase (MTG). Extra‐cellular activation of MTG by the transglutaminase‐activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross‐linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N‐terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N‐terminally shortened variants clearly indicated the highly conserved Leu40‐Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40‐Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X‐ray crystallography. While no structural data could be obtained for the N‐terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI‐family proteins. Enzymes Chymotrypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/1.html; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/24/77.html; streptogrisin‐A (SGPA), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/80.html; streptogrisin‐B (SGPB), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/81.html; subtilisin BPN’, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/62.html; transglutaminase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/2/13.html; transglutaminase‐activating metalloprotease (TAMP), EC3.4.‐.‐; tri‐/tetrapeptidyl aminopeptidase, EC3.4.11.‐; trypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/4.html. Databases The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

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The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ239–248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases.

Distinct streptomycetes such as Streptomyces mobaraensis produce the protein cross‐linking enzyme transglutaminase. Bioinformatic analysis predicted the occurrence of seven sortases exerting transpeptidation reactions similarly to transglutaminase. Here, we report the production and characterization of sortase E2 (Sm‐SrtE2) solubilized by removal of its membrane anchor domain. Sm‐SrtE2 activity was measured using pentapeptides predicted to be cell wall sorting signals of putative sortase substrate proteins. Preferred linkage to Gly3 by Sm‐SrtE2 was in the order LAETG>>LAHTG>>LAQTG~LANTG>LARTG. Chaplin 1 from S. mobaraensis was further demonstrated to be an excellent substrate of both the intrinsic Sm‐SrtE2 and transglutaminase. The unexpected discovery showing Gln‐62 and Gln‐65 of Δ1–50‐Sm‐SrtE2 as transglutaminase cross‐linking sites suggests that low enzyme stability might be due to anchor domain truncation and a disordered N terminus.