Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

A mycobacteriophage-specific repressor with the enhanced operator DNA binding activity at 32°C and no activity at 42°C has not been generated yet though it has potential in developing a temperature-controlled expression vector for mycobacterial system. To create such an invaluable repressor, here we have characterized four substitution mutants of mycobacteriophage L1 repressor by various probes. The W69C repressor mutant displayed no operator DNA binding activity, whereas, P131L repressor mutant exhibited very little DNA binding at 32°C. In contrast, both E36K and E39Q repressor mutants showed significantly higher DNA binding activity at 32°C, particularly, under in vivo conditions. Various mutations also had different effects on the structure, stability and the dimerization ability of L1 repressor. While the W69C mutant possessed a distorted tertiary structure, the P131L mutant dimerized poorly in solution at 32°C. Interestingly, both these mutants lost their two-domain structure and aggregated rapidly at 42°C. Of the native and mutant L1 repressor proteins, W69C and E36K mutants appeared to be the least stable at 32°C. Studies together suggest that the mutants, particularly P131L and E39Q mutants, could be used for creating a high affinity temperature-sensitive repressor in the future.

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